Reprogramming of somatic cells

ABSTRACT

The disclosure relates to a method of reprogramming one or more somatic cells, e.g., partially differentiated or fully/terminally differentiated somatic cells, to a less differentiated state, e.g., a pluripotent or multipotent state. In further embodiments the invention also relates to reprogrammed somatic cells produced by methods of the invention, to uses of said cells, and to methods for identifying agents useful for reprogramming somatic cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/595,041, filed Aug. 25, 2010, which is a national stage filing under35 U.S.C. 371 of International Application PCT/US2008/004516, filed Apr.7, 2008, which claims the benefit of U.S. Provisional Application No.61/036,065, filed Mar. 12, 2008; U.S. Provisional Application No.60/959,341, filed Jul. 12, 2007; and U.S. Provisional Application No.60/922,121, filed Apr. 7, 2007. The entire contents of theafore-mentioned applications are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by grants5-R01-HDO45022, 5-R37-CA084198 and 5-RO1-CA087869 from the NationalInstitutes of Health. The U.S. Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Embryonic development and cellular differentiation are consideredunidirectional pathways because cells undergo a progressive loss ofdevelopmental potency during cell fate specification. Two categories ofpluripotent stem cells are known to date: embryonic stem cells andembryonic germ cells. Embryonic stem cells are pluripotent stem cellsthat are derived directly from an embryo. Embryonic germ cells arepluripotent stem cells that are derived directly from the fetal tissueof aborted fetuses. For purposes of simplicity, embryonic stem cells andembryonic germ cells will be collectively referred to as “ES” cellsherein.

The success of somatic cell nuclear transfer (SCNT) experiments inmammalian species provided proof that the epigenetic state of adultdifferentiated cells is not fixed but remains pliable for reprogrammingby factors present in the oocyte cytoplasm (Byrne et al., 2007; Jaenischand Young, 2008; Wakayama and Yanagimachi, 2001). However, theinefficiency and ethical concerns associated with attempting to clonehuman somatic cells have spurred the field to search for alternativemethods to achieve nuclear reprogramming without using oocytes (Jaenischand Young, 2008). Indeed, fusion of somatic cells to embryonic carcinomacells or embryonic stem (ES) cells results in epigenetic resetting ofthe somatic genome but involves the generation of 4N pluripotent cells,limiting the potential therapeutic use of such cells (Cowan et al.,2005; Tada et al., 2001).

Nevertheless, the reprogramming of somatic cells by fusion with ES cellssuggested that ES cells, similar to the oocyte cytoplasm, containfactors that can induce nuclear reprogramming. An important breakthroughwas achieved by Yamanaka and colleagues, who succeeded in directlyreprogramming fibroblasts into induced pluripotent stem (iPS) cells bytransduction of the four transcription factors Oct4, Sox2, Klf4 andc-Myc (Takahashi and Yamanaka, 2006). Although the initially obtainediPS cells were not normal, several groups have since advanced the directreprogramming technique by generating iPS cells that are epigeneticallyand developmentally indistinguishable from embryo-derived ES cells(Maherali, 2007; Meissner et al., 2007; Okita et al., 2007; Wernig etal., 2007). Moreover, transgenic expression of c-Myc was found to bedispensable for reprogramming, though it accelerated and enhanced theefficiency of reprogramming (Nakagawa et al., 2008; Wernig et al.,2008). Finally, it has also been shown that human iPS cells can begenerated by transduction of defined factors into somatic cells (Park atal., 2008; Takahashi et al., 2007; Yu et al., 2007).

Despite the work that has been done to date, it remains unknown whetherterminally differentiated cells can be reprogrammed to pluripotency withdefined factors, or whether only less differentiated cells such assomatic stem cells can undergo nuclear reprogramming to pluripotency.Moreover, it is unclear whether progressive differentiation of the donorcells affects the efficiency of in vitro reprogramming.

SUMMARY OF THE INVENTION

The present invention provides engineered somatic cells, in which one ormore endogenous pluripotency gene(s) is operably linked to a selectablemarker in such a manner that the expression of the selectable markersubstantially matches the expression of the endogenous pluripotency geneto which the marker is linked. The invention also provides transgenicmice containing these engineered somatic cells.

The present invention also provides methods for reprogramming somaticcells to a less differentiated state. In certain of the methods,engineered somatic cells of the invention are treated with an agent.Cells that express the selectable marker are then selected, and assessedfor pluripotency characteristics. The treatment with an agent may becontacting the cells with an agent which alters chromatin structure, ormay be transfecting the cells with at least one pluripotency gene, orboth.

The present invention further provides methods for identifying an agentthat reprograms somatic cells to a less differentiated state. In certainof the methods, the engineered somatic cells described above arecontacted with a candidate agent. Cells that express the selectablemarker are then selected, and assessed for pluripotency characteristics.The presence of at least a subset of pluripotency characteristicsindicates that the agent is capable of reprogramming somatic cells to aless-differentiated state. The agents identified by the presentinvention can then by used to reprogram somatic cells by contactingsomatic cells with the agents.

The present invention also provides methods for identifying a gene thatcauses the expression of at least one endogenous pluripotency gene insomatic cells. In certain of the methods, the engineered somatic cellsare transfected with a cDNA library prepared from a pluripotent cell,such as an ES cell. The cells that express the appropriate selectablemarker are then selected, and the expression of the appropriateendogenous pluripotency gene is examined. The expression of anendogenous pluripotency gene indicates that the cDNA encodes a proteinwhose expression in the cell results in, directly or indirectly,expression of the endogenous pluripotency gene.

The invention provides methods of deriving reprogrammed somatic cellsfrom somatic cells that have not been genetically modified. Theinvention provides methods of deriving reprogrammed somatic cellswithout use of genetic selection or, in some embodiments, without use ofchemical selection. Reprogrammed somatic cells are derived fromnon-engineered somatic cells according to the invention by, for example,introducing reprogramming agents into non-engineered somatic cellsand/or expressing such agents therein and selecting reprogrammed cellsby any of a variety of methods that do not require presence of exogenousgenetic material within the cells.

In some embodiments, the methods employ morphological criteria toidentify reprogrammed somatic cells from among a population of somaticcells that are not reprogrammed. In some embodiments, the methods employmorphological criteria to identify somatic cells that have beenreprogrammed to an ES-like state from among a population of cells thatare not reprogrammed or are only partly reprogrammed to an ES-likestate.

In some embodiments, the methods employ complement-mediated lysis toeliminate at least some non-reprogrammed somatic cells from a populationof cells that contains at least some reprogrammed somatic cells.

The present invention further provides methods for treating a conditionin an individual in need of such treatment. In certain embodiments,somatic cells are obtained from the individual and reprogrammed by themethods of the invention under conditions suitable for the cells todevelop into cells of a desired cell type. The reprogrammed cells of adesired cell type are then harvested and introduced into the individualto treat the condition. In certain further embodiments, the somaticcells obtained from the individual contain a mutation in one or moregenes. In these instances, in certain embodiments the methods aremodified so that the somatic cells obtained from the individual arefirst treated to restore the one or more normal gene(s) to the cellssuch that the resulting cells carry the normal endogenous gene, whichare then introduced into the individual.

In certain further embodiments, the somatic cells obtained from theindividual are engineered to express one or more genes following theirremoval from the individual. The cells may be engineered by introducinga gene or expression cassette comprising a gene into the cells. The geneor a portion thereof may be flanked by sites for a site-specificrecombinase.

The gene may be one that is useful for purposes of identifying,selecting, and/or generating a reprogrammed cell. In certain embodimentsthe gene encodes an expression product that causes a reduction in DNAmethylation in the cell. For example, the gene may encode an RNA thatinterferes with expression of a DNA methyltransferase, e.g., DNAmethyltransferase 1, 3a, or 3b (Dnmt1, 3a, 3b). The RNA may be a shorthairpin RNA (shRNA) or microRNA precursor. In certain embodiments theRNA is a precursor that is processed intracellularly to yield a shortinterfering RNA (siRNA) or microRNA (miRNA) that inhibits expression ofDnmt1, 3a, or 3b. In certain embodiments the gene encodes a marker thatis usable for positive and for negative selection.

In certain embodiments the gene is one that contributes to initiatingand/or maintaining the reprogrammed state. In certain embodiments thegene is one whose expression product contributes to initiating thereprogrammed state (and in certain embodiments is necessary formaintaining the reprogrammed state) but which is dispensable formaintaining the reprogrammed state. In these instances, in certainembodiments the methods include a step of treating the engineered cellsafter reprogramming in order to reduce or eliminate expression of thegene. In methods in which the reprogrammed cells are differentiated invitro or in vivo after reprogramming, the treatment to reduce oreliminate expression of the gene may occur before or after thereprogrammed cells differentiate. The treatment may comprise causingexcision of at least a portion of the introduced gene, e.g., byintroducing or expressing a recombinase in the cells. In certainembodiments the gene is one whose expression product contributes tomaintaining the reprogrammed state (and in certain embodiments isnecessary for maintaining the reprogrammed state) but which isdispensable once the reprogrammed cells have differentiated into adesired cell type. In these embodiments the methods may include a stepof treating the engineered reprogrammed cells after theirdifferentiation so as to reduce or eliminate expression of the gene.

In certain other embodiments, methods of the invention can be used totreat individuals in need of a functional organ. In the methods, somaticcells are obtained from an individual in need of a functional organ, andreprogrammed by the methods of the invention to produce reprogrammedsomatic cells. Such reprogrammed somatic cells are then cultured underconditions suitable for development of the reprogrammed somatic cellsinto a desired organ, which is than introduced into the individual. Themethods are useful for treating any one of the following conditions: aneurological, endocrine, structural, skeletal, vascular, urinary,digestive, integumentary, blood, autoimmune, inflammatory, or muscularcondition.

The present invention also provides methods for producing a clonedanimal. In the methods, a somatic cell is isolated from an animal havingdesired characteristics, and reprogrammed using the methods of theinvention to produce one or more reprogrammed pluripotent somatic cell(“RPSC”). The RPSCs are then inserted into a recipient embryo, and theresulting embryo is cultured to produce an embryo of suitable size forimplantation into a recipient female, which is then transferred into arecipient female to produce a pregnant female. The pregnant female ismaintained under conditions appropriate for carrying the embryo to termto produce chimeric animal progeny, which is then bred with a wild typeanimal to produce a cloned animal.

In certain embodiments, the RPSCs may alternatively be cryopreserved forfuture cloning uses. In certain other embodiments, genetic modification,such as a targeted mutation, may be introduced into the RPSCs prior toits insertion into a recipient embryo.

The present invention also provides methods for producing a clonedavian. In the methods, a somatic cell is isolated from an avian havingdesired characteristics, and reprogrammed using the methods of theinvention to produce one or more reprogrammed pluripotent somatic cell(“RPSC”). The RPSCs are then inserted into eggs that are unable todevelop into an embryo, and the resulting eggs are then incubated toproduce avian offspring having the genotype of the RPSC, therebyproducing a cloned avian.

It is contemplated that all embodiments described above are applicableto all different aspects of the invention. It is also contemplated thatany of the above embodiments can be freely combined with one or moreother such embodiments whenever appropriate.

As described herein, transgenic and inducible expression of fourtranscription factors (Oct4, Sox2, Klf4, and c-Myc) was used toreprogram mouse B lymphocytes. These factors were sufficient to convertnon-terminally differentiated B cells that have undergone partial B cellreceptor rearrangements to a pluripotent state. Reprogramming of matureB cells required additional ectopic expression of a myeloidtranscription factor CCAAT/enhancer-binding-protein-α (C/EBPα), knownfor its ability to interrupt the transcriptional state maintaining Bcell identity. Multiple iPS lines were clonally derived from bothnon-fully and fully differentiated mature B lymphocytes, and gave riseto adult chimeras, to late term embryos when injected into tetraploidblastocysts, and contributed to the germline. Work described hereinprovides definitive proof for the direct nuclear reprogramming ofterminally differentiated adult cells to pluripotency.

Accordingly, in one embodiment the invention relates to a method ofreprogramming a differentiated somatic cell to a pluripotent state,comprising the steps of contacting a differentiated somatic cell with atleast one reprogramming agent that contributes to reprogramming of saidcell to a pluripotent state; maintaining said cell under conditionsappropriate for proliferation of the cell and for activity of the atleast one reprogramming agent for a period of time sufficient to beginreprogramming of the cell; and functionally inactivating the at leastone reprogramming agent.

In another embodiment the invention relates to a method of reprogramminga differentiated somatic cell to a pluripotent state, comprising thesteps of providing a differentiated somatic cell that contains at leastone exogenously introduced factor that contributes to reprogramming ofsaid cell to a pluripotent state; maintaining the cell under conditionsappropriate for proliferation of the cell and for activity of the atleast one exogenously introduced factor for a period of time sufficientto activate at least one endogenous pluripotency gene; and functionallyinactivating the at least one exogenously introduced factor.

In a further embodiment the invention pertains to a method of selectinga differentiated somatic cell that has been reprogrammed to apluripotent state, comprising the steps of providing a differentiatedsomatic cell that contains at least one exogenously introduced factorthat contributes to reprogramming of the cell to a pluripotent state;maintaining the cell under conditions appropriate for proliferation ofthe cell and for activity of the at least one exogenously introducedfactor for a period of time sufficient to activate at least oneendogenous pluripotency gene; functionally inactivating the at least oneexogenously introduced factor; and differentiating or distinguishingbetween cells which display one or more markers of pluripotency andcells which do not. In one embodiment differentiating or distinguishingbetween cells which display one or more markers of pluripotency andcells which do not comprises selection or enrichment for cellsdisplaying one or more markers of pluripotency and/or selection againstcells which do not display one or more markers of pluripotency.

In some embodiments of the invention the differentiated somatic cell ispartially differentiated. In other embodiments of the invention thedifferentiated somatic cell is fully differentiated.

In some embodiments of the invention the differentiated somatic cell iscell of hematopoetic lineage; in some embodiments the differentiatedsomatic cell is obtained from peripheral blood. In one embodiment of theinvention the differentiated somatic cell is an immune system cell. Inone embodiment the differentiated somatic cell is a macrophage. In oneembodiment the differentiated somatic cell is a lymphoid cell. In otherembodiments of the invention the differentiated somatic cell is a Bcell, such as an immature (e.g., pro-B cell or pre-B cell) or mature(e.g., non-naïve) B-cell.

In some embodiments of the invention the at least one exogenouslyintroduced factor is a polynucleotide. In other embodiments the at leastone exogenously introduced factor is a polypeptide. In one embodimentthe at least one exogenously introduced factor is selected from thegroup consisting of Oct4, Sox2, Klf-4, Nanog, Lin28, c-Myc andcombinations thereof. In particular embodiments of the invention thedifferentiated somatic cell contains exogenously introduced Oct4, Sox2,and Klf-4 exogenously introduced Oct4, Sox2, Klf-4 and c-Myc.

In one embodiment of the invention the at least one exogenouslyintroduced factor is selected from the group consisting of Oct4, Sox2,Klf-4, c-Myc and combinations thereof and the differentiated somaticcell further contains at least one exogenously introduced factor (e.g.,a polynucleotide or polypeptide) capable of inducing dedifferentiationof the differentiated somatic cell. In some embodiments the factorcapable of inducing dedifferentiation of said differentiated somaticcell is selected from the group consisting of at least onepolynucleotide which downregulates B cell late specific markers, atleast one polynucleotide which inhibits expression of Pax5, at least onepolypeptide which downregulates B cell late specific markers, at leastone polypeptide which inhibits expression of Pax5, and combinationsthereof. In one embodiment of the invention the factor capable ofinducing dedifferentiation of said differentiated somatic cell is C/EBPαor a human homolog of C/EBPα.

In particular embodiments of the invention the at least one exogenouslyintroduced factor is introduced using a vector, e.g., an induciblevector or a conditionally expressed vector. In one aspect the at leastone exogenously introduced factor is introduced using a vector which isnot subject to methylation-mediated silencing. In yet another embodimentthe at least one exogenously introduced factor is introduced using aviral vector such as a retroviral or lentiviral vector.

In one embodiment of the invention the differentiated somatic cell ismaintained in the presence of hematopoetic cytokines and growth factorsor is cultured on media comprising bone marrow stromal cells.

In some embodiments of the present invention the endogenous pluripotencygene is selected from the group consisting of Nanog, Oct4, Sox2 andcombinations thereof. In other embodiments the endogenous pluripotencygene is co-expressed with a selectable marker, such as an antibioticresistance gene or luminescent marker. In particular embodiments thedifferentiated somatic cell further comprises at least onepolynucleotide encoding a selectable marker operably linked toexpression control elements that regulate expression of said at leastone endogenous pluripotency gene. In specific embodiments thedifferentiated somatic cell comprises a selectable gene in the Oct4locus, the Nanog locus, or both the Oct4 and Nanog loci. In a certainembodiment the at least one exogenously introduced factor is introducedusing an inducible vector and wherein functionally inactivating said atleast one exogenously introduced factor comprises rendering theconditions under which said cell is maintained unsuitable for inducibleexpression of said vector.

In some embodiments of the invention, markers of pluripotency areselected from the group consisting of expression of a pluripotency gene,expression of a gene whose expression is a direct or indirect result ofexpression of a pluripotency gene, expression of alkaline phosphatase,expression of SSEA1, expression of SSEA3, expression of SSEA4,expression of TRAF-60, expression of Nanog, expression of Oct4,expression of Fxb15, morphology characteristic of an ES cell or an EScell colony, ability to participate in formation of chimeras thatsurvive to term, ability to differentiate into cells havingcharacteristics of endoderm, mesoderm and ectoderm when injected intoSCID mice, presence of two active X chromosomes, resistance to DNAmethylation, and combinations thereof.

The invention also relates to an isolated pluripotent cell derived froma reprogrammed differentiated somatic cell in accordance with methods ofthe invention. In particular the invention relates to a purifiedpopulation of somatic cells comprising at least 70% pluripotent cellsderived from reprogrammed differentiated somatic cells.

The invention further relates to an isolated pluripotent cell producedby a method comprising (a) providing a differentiated somatic cell thatcontains at least one exogenously introduced factor that contributes toreprogramming of said cell to a pluripotent state; (b) maintaining saidcell under conditions appropriate for proliferation of said cell and foractivity of said at least one exogenously introduced factor for a periodof time sufficient to activate at least one endogenous pluripotencygene; (c) functionally inactivating said at least one exogenouslyintroduced factor; and (d) differentiating cells which display one ormore markers of pluripotency from cells which do not.

The invention also relates to a purified population of somatic cellscomprising at least 70% pluripotent cells derived from reprogrammeddifferentiated somatic cells produced by a method comprising (a)providing a differentiated somatic cell that contains at least oneexogenously introduced factor that contributes to reprogramming of saidcell to a pluripotent state; (b) maintaining said cell under conditionsappropriate for proliferation of said cell and for activity of said atleast one exogenously introduced factor for a period of time sufficientbegin reprogramming of said cell or to activate at least one endogenouspluripotency gene; (c) functionally inactivating said at least oneexogenously introduced factor; and (d) differentiating cells whichdisplay one or more markers of pluripotency and cells which do not.

In another aspect the invention relates to a method of producing apluripotent cell from a somatic cell, comprising the steps of (a)providing one or more somatic cells that each contain at least oneexogenously introduced factor that contributes to reprogramming of saidcell to a pluripotent state, wherein said exogenously introduced factoris introduced using an inducible vector which is not subject tomethylation-induced silencing; (b) maintaining said one or more cellsunder conditions appropriate for proliferation of said cells and foractivity of said at least one exogenously introduced factor for a periodof time sufficient begin reprogramming of said cell or to activate atleast one endogenous pluripotency gene; (c) functionally inactivatingsaid at least one exogenously introduced factor; (d) selecting one ormore cells which display a marker of pluripotency; (e) generating achimeric embryo utilizing said one or more cells which display a markerof pluripotency; (f) obtaining one or more somatic cells from saidchimeric embryo; (g) maintaining said one or more somatic cells underconditions appropriate for proliferation of said cells and for activityof said at least one exogenously introduced factor for a period of timesufficient to begin reprogramming said cell or to activate at least oneendogenous pluripotency gene; and (h) differentiating between cellswhich display one or more markers of pluripotency and cells which donot. In a particular embodiment the method yields a purified populationof somatic cells comprising at least 70% pluripotent cells derived fromreprogrammed differentiated somatic cells

The invention also relates to an isolated pluripotent cell produced by amethod comprising (a) providing one or more somatic cells that eachcontain at least one exogenously introduced factor that contributes toreprogramming of said cell to a pluripotent state, wherein saidexogenously introduced factor is introduced using an inducible vectorwhich is not subject to methylation-induced silencing; (b) maintainingsaid one or more cells under conditions appropriate for proliferation ofsaid cells and for activity of said at least one exogenously introducedfactor for a period of time sufficient to begin reprogramming said cellor to activate at least one endogenous pluripotency gene; (c)functionally inactivating said at least one exogenously introducedfactor; (d) selecting one or more cells which display a marker ofpluripotency; (e) generating a chimeric embryo utilizing said one ormore cells which display a marker of pluripotency; (f) obtaining one ormore somatic cells from said chimeric embryo; (g) maintaining said oneor more somatic cells under conditions appropriate for proliferation ofsaid cells and for activity of said at least one exogenously introducedfactor for a period of time sufficient to activate at least oneendogenous pluripotency gene; and (h) differentiating cells whichdisplay one or more markers of pluripotency and cells which do not.

In preferred embodiments of the invention the methods yield a purifiedpopulation of somatic cells comprising at least 70% (e.g., 70%, 75%,80%, 85%, 90%, 95%, 99%) pluripotent cells derived from reprogrammeddifferentiated somatic cells. In particular embodiments the pluripotentcells are genetically homogenous.

The invention also pertains to a purified population of somatic cellscomprising at least 70% pluripotent cells derived from reprogrammeddifferentiated somatic cells produced by a method comprising (a)providing one or more somatic cells that each contain at least oneexogenously introduced factor that contributes to reprogramming of saidcell to a pluripotent state, wherein said exogenously introduced factoris introduced using an inducible vector which is not subject tomethylation-induced silencing; (b) maintaining said one or more cellsunder conditions appropriate for proliferation of said cells and foractivity of said at least one exogenously introduced factor for a periodof time sufficient to begin reprogramming of said cell or to activate atleast one endogenous pluripotency gene; (c) functionally inactivatingsaid at least one exogenously introduced factor; (d) selecting one ormore cells which display a marker of pluripotency; (e) generating achimeric embryo utilizing said one or more cells which display a markerof pluripotency; (f) obtaining one or more somatic cells from saidchimeric embryo; (g) maintaining said one or more somatic cells underconditions appropriate for proliferation of said cells and for activityof said at least one exogenously introduced factor for a period of timesufficient to begin reprogramming said cell or to activate at least oneendogenous pluripotency gene; and (h) differentiating cells whichdisplay one or more markers of pluripotency and cells which do not.

The invention also encompasses a method of reprogramming adifferentiated immune cell to a pluripotent state, comprising the stepsof (a) providing a differentiated immune cell that contains exogenouslyintroduced Oct4, Sox2, Klf-4 and c-Myc, each under the control of aninducible vector, and further contains exogenously introduced C/EBPα;(b) maintaining said cell under conditions appropriate for proliferationof said cell and for activity of Oct4, Sox2, Klf-4, c-Myc and C/EBPα fora period of time sufficient to activate endogenous Nanog and/or Oct4;and (c) functionally inactivating exogenously introduced Oct4, Sox2,Klf-4 and c-Myc. In one embodiment of the method said inducible vectoris not subject to methylation-derived silencing.

The invention also relates to a purified population of immune cellscomprising at least 70% pluripotent cells derived from reprogrammeddifferentiated immune cells produced by a method comprising the steps of(a) providing a differentiated immune cell that contains exogenouslyintroduced Oct4, Sox2, Klf-4 and c-Myc, each under the control of aninducible vector, and further contains exogenously introduced C/EBPα;(b) maintaining said cell under conditions appropriate for proliferationof said cell and for activity of Oct4, Sox2, Klf-4, c-Myc and C/EBPα fora period of time sufficient to activate endogenous Nanog and/or Oct4;and (c) functionally inactivating exogenously introduced Oct4, Sox2,Klf-4 and c-Myc.

The invention also relates to a method of identifying a reprogrammingagent comprising (a) providing one or more somatic cells that eachcontain at least one exogenously introduced factor that contributes toreprogramming of said cell to a pluripotent state, wherein each of saidexogenously introduced factors is introduced using an inducible vectorwhich is not subject to methylation-induced silencing and the expressionof which is controlled by regulatory elements induced by distinctinducers; (b) maintaining said one or more cells under conditionsappropriate for proliferation of said cells and for activity of said atleast one exogenously introduced factor for a period of time sufficientto reprogram said cell or to activate at least one endogenouspluripotency gene; (c) functionally inactivating said at least oneexogenously introduced factor; (d) selecting one or more cells whichdisplay a marker of pluripotency; (e) generating a chimeric embryoutilizing said one or more cells which display a marker of pluripotency;(f) obtaining one or more somatic cells from said chimeric embryo; (g)maintaining said one or more somatic cells under conditions appropriatefor proliferation of said cells and for activity of said at least oneexogenously introduced factor wherein activity of said at least oneexogenously introduced factor is insufficient by itself to activate atleast one endogenous pluripotency gene; (h) contacting the somatic cellof (g) with one or more candidate reprogramming agents; and (i)identifying cells contacted with said one or more candidatereprogramming agents which display one or more markers of pluripotency,wherein candidate reprogramming agents which induce the somatic cell of(g) to display one or more markers of pluripotency are identified asreprogramming agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of an inducible Oct4 allele. Thefirst integration vector, inducible Oct4 integration vector, contains anOct4 gene driven by a tetracycline-inducible promoter (Tet-Op). TheTet-Op-Oct4 cassette is flanked by a splice-acceptor double poly-Asignal (SA-dpA) at its 5′ end and a SV40 polyA tail (SV40-pA) at its 3′end. The second integration vector, tetracycline activator integrationvector, contains a mutant form of tetracycline activator, M2-rtTA, whichis more responsive to doxycycline (Dox) induction than the wild typeactivator (Urlinger at al., Prod Natl Acad Sci USA 97(14):7963 2000)).

FIGS. 2A-2B show the generation of Oct4- and Nanog-selected iPS cells.As illustrated in FIG. 2A, an IRES-GfpNeo fusion cassette was insertedinto the BclI site downstream of Oct4 exon 5. Correctly targeted ES cellclones were screened by Southern analysis of NcoI digested DNA using a5′ external probe. The Nanog gene was targeted as described in Mitsui etal., Cell 123(5):631 (2003). FIG. 2B shows the total number (left scale)and percentages (right scale) of AP- and strong SSEA1-positive coloniesof Oct4- and Nanogneo MEFs 4 weeks after infection and neo selection.

FIG. 3 shows the transgenic inducible expression of OCT4, Sox2, Klf4 andc-Myc in the mouse B cell lineage, in particular a schematic drawingrepresenting the strategy used in this study for reprogramming cellsfrom the B cell lineage.

FIG. 4 shows a schematic representation of experiments attempting tomeasure reprogramming efficiency. 3*10^(^6) CD19+ adult B cells wereinfected with retrovirus encoding C/EBPα-NeoR construct, and after 24hours we sorted IgM+IgD+ mature adult B cells and plated them as singlecells in 96-well plates preplated with OP9 stromal cell line. Cells weregrown in conditioned medium+Dox+LIF throughout the experiment. On day 6,culture wells were subjected to puromycin and neomycin selections for 5days, which allowed only the growth of transgenic B cells infected withC/EBPα. On day 20, the wells containing drug resistant cells werescreened for Nanog-GFP expression by FACS analysis. Wells that scoredpositive were subsequently passaged on MEFs in ES media and grown intoiPS cell lines.

DETAILED DESCRIPTION OF THE INVENTION

Nuclear reprogramming, which pertains to the concept of rewiring theepigenetic state of a somatic nucleus to that of another cell type, canbe achieved by different approaches. The most recently establishedstrategy to reprogram somatic cells to pluripotency involves directectopic expression of defined transcription factors in somatic cells(Okita at al., 2007; Takahashi and Yamanaka, 2006; Wernig et al., 2007).This enforced factor expression appears to initiate a sequence ofstochastic events occurring over a relatively extended time period inculture that eventually leads to generation of a small fraction of cellsthat have acquired a stable pluripotent state (Jaenisch and Young,2008). The transduced factors are required for an initial period of timein the reprogramming process (Brambrink at al., 2008; Stadtfeld et al.,2008), during which they may interact with endogenous pluripotency genes(Boyer et al., 2005; Loh at al., 2006) and gradually induce epigeneticchanges that subsequently sustain a stable epigenetic state that isindistinguishable from that of inner cell mass-derived ES cells. Duringthis process, the de novo methyltransferases Dnmt3a and Dnmt3b alsobecome activated and in turn methylate and silence the virallytransduced factors. Silencing of the exogenous factors is crucial forsubsequent differentiation of the iPS cells (Brambrink at al., 2008;Takahashi et al., 2007; Wernig at al., 2007; Stadfeld et al., 2008).

In the development of cells along the B cell lineage, sequentialintrinsic genetic DNA rearrangements in the heavy and light chainimmunoglobulin loci genetically mark the different consecutive stages ofB cell maturation (Jung et al., 2006). Cells at the Pro-B stage ofdevelopment initiate immunoglobulin rearrangements, a process involvingthe assembly of V (variable), D (diversity) and J (joining) genesegments. Assembly of the heavy chain locus (IgH) precedes that of thelight chain loci (IgL) (Jung et al., 2006). In addition, therearrangements of the IgH locus are sequential, with D_(H) to J_(H)joining occurring on both alleles prior to V_(H) to D_(H)J_(H) segmentrearrangement (Papavasiliou et al., 1997). The productive assembly ofV_(H)-D_(H)J_(H) variable gene region indirectly signals differentiationto the next stage, in which IgL chains are assembled with Igκrearrangement generally preceding that of Igλ (Papavasiliou et al.,1997). Productive IgL chain generation eventually leads to theassociation of functional light and heavy chain proteins, which togetherform the B cell receptor on the cell surface. The resulting B cells canmigrate to the periphery where, upon encountering a cognate antigen,they can exert proper immunological functions (Schlissel, 2003).

Work described herein used cells from this highly ordered developmentalpathway that carry distinct, sequentially-acquired, genetic“fingerprints” that would allow accurate retrospective assessment of thedevelopmental stage of the donor B cell nucleus that was able togenerate the respective monoclonal iPS line. In particular, as describedherein, iPS cells were generated from pro- and pre-B cells bytransduction with the reprogramming factors Oct4, Sox2, c-Myc and Klf4and from mature B cells by the additional over expression of C/EBPα, awell-characterized myeloid transcription factor. This work shows thatthe reprogrammed cells carried the genetic rearrangements characteristicof donor non-terminally differentiated and mature terminallydifferentiated B cells and were able to generate adult chimeric mice andcontribute to germline. These results indicate that specificcombinations of reprogramming factors can reset the genome of terminallydifferentiated cells to a pluripotent state.

The work described herein provides conclusive evidence that terminallydifferentiated mature B cells obtained from adult mice can be directlyreprogrammed into ES-like cells in vitro. The donor B cell populationthat eventually underwent successful reprogramming had completed acomplex differentiation pathway involving epigenetic and geneticchanges: an initial commitment to the hematopoietic and subsequently tothe B cell lineage; acquisition of productive heavy and light chainrearrangements; egression from the bone marrow to repopulate peripherallymphoid organs in adult mice, and as observed in one of the cell linesobtained, acquisition of somatic hypermutations in variable region of Bcell receptor genes in response to antigen stimuli. Thus, robust ectopicexpression of Oct4, Sox2, Klf-4, c-Myc and C/EBPα transcription factorsinduced reprogramming of fully differentiated lymphoid cells topluripotency with a relatively high efficiency of ˜1 in 30 cells.

Importantly, results described herein demonstrate that under similarinduction levels of Oct4, Sox2, Klf4 and c-Myc transgenes in the B celllineage, non-terminally differentiated and terminally differentiated Bcells respond differently to these factors. Robust reprogramming offully differentiated mature B lymphocytes to pluripotency was achievedwhen the C/EBPα transcription factor, which normally plays a role ingranulocyte cell fate specification, was initially over-expressed (Ramjiand Foka, 2002). Thomas Graf and colleagues (Xie at al., 2004) haveshown that overexpression of C/EBPα converted B cells intomacrophage-like cells by downregulating B cell late specific markers(e.g., CD19) through inhibition of Pax5 functions and facilitatingextinction of the early B cell regulators, EBF1 and E2A transcriptionfactors. In addition, C/EBPα induced up-regulation of components of amyeloid transcriptional network (Laiosa et al., 2006; Xie et al., 2004).These observations are relevant for understanding the mechanisms ofreprogramming and suggest a crucial role for C/EBPα in inducing thereprogramming process of mouse mature B lymphocytes. This suggests anumber of mutually non-exclusive possibilities:

1) C/EBPα may cross-antagonize key regulators of the B celltranscriptional network that maintain the mature B cell identity. Thismay facilitate the dedifferentiation of B cells to a less differentiatedstate, allowing Oct4, Sox2, Klf4 and c-Myc transgene-inducedreprogramming. This explanation is consistent with observations that thedifferentiation state of the donor cells is known to influence theefficiency of reprogramming by nuclear transplantation, as neural andkeratinocyte stem cells were more efficiently reprogrammed than othermore differentiated cells obtained from the same lineage (Blelloch etal., 2006; Li at al., 2007). As conditional deletion of Pax5 in mature Bcells resulted in their dedifferentiation and loss of several mature Bcell markers (Cobaleda at al., 2007a), it may be that deletion of Pax5would also sensitize mature B cells to reprogramming to pluripotency byOct4, Sox2, Klf4 and c-Myc.

2) C/EBPα may convert mature B cells into macrophage-like cells (Xie atal., 2004) which have a different epigenetic state that possibly allowsenhanced accessibility to target genes of Oct4, Sox2, Klf4, and/or c-Mycthat would facilitate the efficient induction of the endogenousauto-regulatory loop governing the pluripotent state (Boyer at al.,2005; Loh et al., 2006).

3) C/EBPα-mediated overexpression may enable mature B cells totransition from a state of growing in suspension to become adherentcells in the presence of OP9 cells, which might be a rate-limiting eventin their reprogramming.

4) Finally, other combinations of factors than those used in theexamples may be able to reprogram mature B lymphocytes under differentculture conditions.

Applicants have devised novel methods of reprogramming somatic cells,e.g., partially or fully differentiated somatic cells, to generatepluripotent cells or multipotent cells. It should be noted that themethods of the invention are not intended to encompass prior art methodsincluding, but not limited to, somatic cell nuclear transfer. That is,it is not within the scope of the invention to reprogram a somatic cellby contacting the nucleus of said cell with the intact cytoplasm of anoocyte, i.e., by transferring the nucleus of said cell into anenucleated oocyte. While some embodiments of the invention encompassmethods of reprogramming a nucleus of a somatic cell which has beenisolated from the cytoplasm in which it is ordinarily contained, andoptionally subsequently transferring said nucleus to an enucleated cellof the same or different cell type, these embodiments do not encompassmethods in which the reprogramming agent is an enucleated oocyte.Applicants have also devised novel methods to identify agents that,alone or in combination with other factors and/or conditions, reprogramsomatic cells.

Certain of the methods of the invention make use of characteristics thatdiffer between ES cells (e.g., ES cells generated using conventionalmethods described in the Background) and somatic cells. Thesecharacteristics distinguish ES cells from somatic cells that have notbeen reprogrammed and are used as a basis to identify reprogrammed cells(induced pluripotent cells) in certain of the methods.

One such characteristic is the increased ability of ES cells to survivedemethylation of genomic DNA relative to somatic cells. Somatic cellsare treated in any of a variety of ways that may result inreprogramming, and the cells are subjected to a procedure that resultsin DNA demethylation. In certain embodiments of the invention, somaticcells that are able to survive the procedure are identified as beingreprogrammed or having an increased likelihood of being reprogrammedrelative to cells which are not able to survive the procedure. Incertain embodiments of the invention a candidate reprogramming agent,e.g., a treatment or factor, that has resulted in at least a portion ofthe cells becoming resistant to DNA demethylation (i.e., able to surviveunder conditions of DNA methylation) is identified as an agent usefulfor reprogramming a somatic cell.

Another characteristic of ES cells that distinguishes them from somaticcells is that ES cells contain two transcriptionally active Xchromosomes, whereas in somatic cells one X chromosome is normallylargely or completely transcriptionally inactive (see Avner, P. andHeard, E., Nature Reviews Genetics, 2: 59-67, 2001; Eggan, K., et al.,Science, 290(5496):1578-81, 2000). According to one embodiment of theinvention, somatic cells are treated in any of a variety of ways thatmay result in reprogramming. The treatment can be, for example,contacting the cells with a candidate reprogramming agent, e.g., atreatment or factor. In certain embodiments of the invention, cells inwhich both X chromosomes are transcriptionally active are identified asreprogrammed or having an increased likelihood of being reprogrammedrelative to cells in which only one X chromosome is transcriptionallyactive. In certain embodiments of the invention a candidatereprogramming agent, e.g., a treatment or factor, that has resulted inat least a portion of the cells having two transcriptionally active Xchromosomes is identified as a treatment useful for reprogramming asomatic cell. In some embodiments, one step of the method involvesselecting for cells that have only one transcriptionally active Xchromosome, and a subsequent step of the method comprises selecting forcells that have activated their inactive X chromosome.

Certain other of the methods take advantage of the engineered somaticcells designed by Applicants, in which an endogenous gene typicallyassociated with pluripotency (“pluripotency gene”) is engineered to beoperably linked to a selectable marker in a manner that the expressionof the endogenous pluripotency gene substantially matches the expressionof the selectable marker. Because pluripotency genes are generallyexpressed only in pluripotent cells and not in somatic cells, theexpression of an endogenous pluripotency gene(s) is an indication ofsuccessful reprogramming. Having a selectable marker operably linked toan endogenous pluripotency gene gives one a powerful mechanism to selectfor potentially reprogrammed somatic cells, which may be a rareoccurrence. The resulting cells may be alternatively or additionallyassessed for other pluripotency characteristics to confirm whether asomatic cell has been successfully reprogrammed to pluripotency.

Accordingly, in one embodiment the invention relates to a method ofreprogramming one or more somatic cells, e.g., partially differentiatedor fully/terminally differentiated somatic cells, to a lessdifferentiated state, e.g., a pluripotent or multipotent state. Ingeneral the method comprises the steps of contacting the somatic cell orisolated somatic cell nucleus with at least one reprogramming agent thatcontributes to reprogramming of the cell to a pluripotent state;maintaining the cell under conditions appropriate for proliferation ofthe cell and for activity of the reprogramming agent for a period oftime sufficient to activate an endogenous pluripotency gene, andfunctionally inactivating the reprogramming agent, e.g., inactivating orremoving the reprogramming agent. In further embodiments the inventionalso relates to reprogrammed somatic cells produced by methods of theinvention and to uses of said cells.

Generating pluripotent or multipotent cells by using the methods of thepresent invention has at least two advantages. First, the methods of thepresent invention allow one to generate autologous pluripotent cells,which are cells specific to a patient. The use of autologous cells incell therapy offers a major advantage over the use of non-autologouscells, which are more likely to be subject to immunological rejection.In contrast, autologous cells are less likely to elicit significantimmunological responses. Second, the methods of the present inventionallow one to generate pluripotent cells without using embryos, oocytesand/or nuclear transfer technology.

TERMINOLOGY

The articles “a”, “an” and “the” as used herein, unless clearlyindicated to the contrary, should be understood to include the pluralreferents. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. It should it be understood that,in general, where the invention, or aspects of the invention, is/arereferred to as comprising particular elements, features, etc., certainembodiments of the invention or aspects of the invention consist, orconsist essentially of, such elements, features, etc. For purposes ofsimplicity those embodiments have not in every case been specificallyset forth in haec verba herein. It should also be understood that anyembodiment of the invention, e.g., any embodiment found within the priorart, can be explicitly excluded from the claims, regardless of whetherthe specific exclusion is recited in the specification. For example, anyagent may be excluded from the set of candidate reprogramming agents,and any gene can be excluded from the set of pluripotency genes.

Where ranges are given herein, the invention includes embodiments inwhich the endpoints are included, embodiments in which both endpointsare excluded, and embodiments in which one endpoint is included and theother is excluded. It should be assumed that both endpoints are includedunless indicated otherwise. Furthermore, it is to be understood thatunless otherwise indicated or otherwise evident from the context andunderstanding of one of skill in the art, values that are expressed asranges can assume any specific value or subrange within the statedranges in different embodiments of the invention, to the tenth of theunit of the lower limit of the range, unless the context clearlydictates otherwise. It is also understood that where a series ofnumerical values is stated herein, the invention includes embodimentsthat relate analogously to any intervening value or range defined by anytwo values in the series, and that the lowest value may be taken as aminimum and the greatest value may be taken as a maximum. Numericalvalues, as used herein, include values expressed as percentages. For anyembodiment of the invention in which a numerical value is prefaced by“about” or “approximately”, the invention includes an embodiment inwhich the exact value is recited. For any embodiment of the invention inwhich a numerical value is not prefaced by “about” or “approximately”,the invention includes an embodiment in which the value is prefaced by“about” or “approximately”. “Approximately” or “about” generallyincludes numbers that fall within a range of 1% or in some embodiments5% of a number in either direction (greater than or less than thenumber) unless otherwise stated or otherwise evident from the context(except where such number would impermissibly exceed 100% of a possiblevalue).

Furthermore, it is to be understood that the invention encompasses allvariations, combinations, and permutations in which one or morelimitations, elements, clauses, descriptive terms, etc., from one ormore of the listed claims is introduced into another claim dependent onthe same base claim (or, as relevant, any other claim) unless otherwiseindicated or unless it would be evident to one of ordinary skill in theart that a contradiction or inconsistency would arise. Where elementsare presented as lists, e.g., in Markush group or similar format, it isto be understood that each subgroup of the elements is also disclosed,and any element(s) can be removed from the group.

Certain claims are presented in dependent form for the sake ofconvenience, but any dependent claim may be rewritten in independentformat to include the limitations of the independent claim and any otherclaim(s) on which such claim depends, and such rewritten claim is to beconsidered equivalent in all respects to the dependent claim (eitheramended or unamended) prior to being rewritten in independent format. Itshould also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one act,the order of the acts of the method is not necessarily limited to theorder in which the acts of the method are recited, but the inventionincludes embodiments in which the order is so limited. It iscontemplated that all embodiments described above are applicable to alldifferent aspects of the invention. It is also contemplated that any ofthe above embodiments can be freely combined with one or more other suchembodiments whenever appropriate.

Somatic Cells

Somatic cells of the invention may be primary cells (non-immortalizedcells), such as those freshly isolated from an animal, or may be derivedfrom a cell line (immortalized cells). The cells may be maintained incell culture following their isolation from a subject. In certainembodiments the cells are passaged once or more than once (e.g., between2-5, 5-10, 10-20, 20-50, 50-100 times, or more) prior to their use in amethod of the invention. In some embodiments the cells will have beenpassaged no more than 1, 2, 5, 10, 20, or 50 times prior to their use ina method of the invention. They may be frozen, thawed, etc. In certainembodiments of the invention the somatic cells are obtained from afemale. The somatic cells used may be native somatic cells, orengineered somatic cells, i.e., somatic cells which have beengenetically altered.

Somatic cells of the present invention are typically mammalian cells,such as, for example, human cells, primate cells or mouse cells. Theymay be obtained by well-known methods and can be obtained from any organor tissue containing live somatic cells, e.g., blood, bone marrow, skin,lung, pancreas, liver, stomach, intestine, heart, reproductive organs,bladder, kidney, urethra and other urinary organs, etc. Mammaliansomatic cells useful in the present invention include, but are notlimited to, sertoli cells, endothelial cells, granulosa epithelial,neurons, pancreatic islet cells, epidermal cells, epithelial cells,hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells,melanocytes, chondrocytes, lymphocytes (B and T lymphocytes),erythrocytes, macrophages, monocytes, mononuclear cells, cardiac musclecells, and other muscle cells, etc. The term “somatic cells”, as usedherein, also includes adult stem cells. An adult stem cell is a cellthat is capable of giving rise to all cell types of a particular tissue.Exemplary adult stem cells include hematopoietic stem cells, neural stemcells, and mesenchymal stem cells.

In some embodiments cells are selected based on their expression of anendogenous marker known to be expressed only or primarily in a desiredcell type. For example, vimentin is a fibroblast marker. Other usefulmarkers include various keratins, cell adhesion molecules such ascadherins, fibronectin, CD molecules, etc. The population of somaticcells may have an average cell cycle time of between 18 and 96 hours,e.g., between 24-48 hours, between 48-72 hours, etc. In someembodiments, at least 90%, 95%, 98%, 99%, or more of the cells would beexpected to divide within a predetermined time such as 24, 48, 72, or 96hours.

Methods of the invention may be used to reprogram one or more somaticcells, e.g., colonies or populations of somatic cells. In someembodiments a population of cells of the present invention issubstantially uniform in that at least 90% of the cells display aphenotype or characteristic of interest. In some embodiments at least95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9, 99.95% or more of the cellsdisplay a phenotype or characteristic of interest. In certainembodiments of the invention the somatic cells have the capacity todivide, i.e., the somatic cells are not post-mitotic. The cells may, forexample, have an average cell cycle time (i.e., time required for a cellto complete a single cell division cycle) of between 18-72 hours, e.g.,between 24-48 hours when maintained in culture under standard cultureconditions known in the art.

Differentiated somatic cells of the invention are partially orcompletely differentiated. Differentiation is the process by which aless specialized cell becomes a more specialized cell type. Celldifferentiation can involve changes in the size, shape, polarity,metabolic activity, gene expression and/or responsiveness to signals ofthe cell. For example, hematopoietic stem cells differentiate to giverise to all the blood cell types including myeloid (monocytes andmacrophages, neutrophils, basophils, eosinophils, erythrocytes,megakaryocytes/platelets, dendritic cells) and lymphoid lineages(T-cells, B-cells, NK-cells). During progression along the path ofdifferentiation, the ultimate fate of a cell becomes more fixed. Asshown by work described herein, both partially differentiated somaticcells (e.g., immature B cells such as pre-B cells and pro-B cells) andfully differentiated somatic cells (e.g., mature B cells, non-naïvemature B cells) can be reprogrammed as described herein to producemultipotent or pluripotent cells (also known as “induced pluripotentcells”).

Reprogramming and Pluripotent Cells

Differentiation status of cells is a continuous spectrum, with aterminally differentiated state at one end of this spectrum andde-differentiated state (pluripotent state) at the other end.Reprogramming, as used herein, refers to a process that alters orreverses the differentiation status of a somatic cell, which can beeither partially or terminally differentiated. Reprogramming includescomplete reversion, as well as partial reversion, of the differentiationstatus of a somatic cell. In other words, the term “reprogramming”, asused herein, encompasses any movement of the differentiation status of acell along the spectrum toward a less-differentiated state. For example,reprogramming includes reversing a multipotent cell back to apluripotent cell, and reversing a terminally differentiated cell back toeither a multipotent cell or a pluripotent cell. In one embodiment,reprogramming of a somatic cell turns the somatic cell all the way backto a pluripotent state. In another embodiment, reprogramming of asomatic cell turns the somatic cell back to a multipotent state. Theterm “less-differentiated state”, as used herein, is thus a relativeterm and includes a completely de-differentiated state and a partiallydifferentiated state.

A pluripotent cell is a cell that has the potential to divide in vitrofor a long period of time (e.g., greater than one year) and has theunique ability to differentiate into cells derived from all threeembryonic germ layers—endoderm, mesoderm and ectoderm. Pluripotent cellshave the potential to differentiate into the full range of daughtercells having distinctly different morphological, cytological orfunctional phenotypes unique to a specific tissue. By contrast,descendants of pluripotent cells are restricted progressively in theirdifferentiation potential, with some cells having only one fate. Amultipotent cell is a cell that is able to differentiate into some butnot all of the cells derived from all three germ layers. Thus, amultipotent cell is a partially differentiated cell. Adult stem cellsare also multipotent or partially differentiated cells. Known adult stemcells include, for example, hematopoietic stem cells and neural stemcells.

Treatment of Somatic Cell(s) with Reprogramming Agent

As described herein, one or more (e.g., a population or colony) somaticcells, e.g., differentiated somatic cells, is treated or contacted withat least one reprogramming agent or factor that contributes toreprogramming of said cell. The terms “contact”, “contacting”, “treat”,“treating”, etc., are used interchangeably herein and include subjectinga cell to any kind of process or condition or performing any kind ofprocedure on the cell. The treatment can be, by way of non-limitingexample, contacting the cells with a known or candidate reprogrammingagent (e.g., an agent which alters the chromatin structure of the cell,an agent which decreases DNA methylation, an agent which decreaseshistone acetylation) transfecting the cells with a polynucleotideencoding a reprogramming agent, and/or culturing the cells underconditions that differ from standard culture conditions in which suchcells are typically maintained. For example, the temperature or pH couldbe varied. Multiple known or candidate reprogramming agents may be usedconcurrently/simultaneously or sequentially. In another embodiment,methods of the invention may further include repeating the steps oftreating the cells with an agent or factor. The agent used in therepeating treatment may be the same as, or different from, the one usedduring the first treatment. Reprogramming agents of the invention can bepolynucleotides, polypeptides, small molecules, etc.

The cells may be contacted with a reprogramming factor or agent forvarying periods of time. In some embodiments the cells are contactedwith the agent for a period of time between 1 hour and 30 days. In someembodiments the cells are contacted with the agent for a period of timesufficient to reprogram the cell or to activate an endogenouspluripotency gene. For example, the period may be 1 day, 5 days, 7 days,10 days, 12 days, 14 days or 20 days. The reprogramming agent may beremoved or inactivated prior to performing a selection to enrich forpluripotent cells or assessing the cells for pluripotencycharacteristics.

According to some embodiments of the invention, after the somaticcell(s) are contacted with the reprogramming agent or factor, they aremaintained under conditions appropriate for proliferation of the celland for activity of the reprogramming agent or factor for a timesufficient to reprogram the cell or to activate at least one endogenouspluripotency gene. Cells may be maintained in culture for varyingperiods of time while reprogramming takes place, prior to selection ofor enrichment for reprogrammed cells. Thus in certain methods, somaticcells which have been contacted with a reprogramming agent or factor aremaintained in culture for more than 3 days prior to identifying orselecting for reprogrammed cells. In some methods, said cells aremaintained in culture for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21 or more days (e.g., between 3-5 weeks) priorto identifying or selecting for reprogrammed cells.

In addition, in particular embodiments of the invention the somaticcells which have been contacted with one or more reprogramming agentsaccording to the described methods are maintained under conditionsappropriate for proliferation of said cells. Conditions appropriate forthe maintenance and proliferation of particular cell types will beapparent to the skilled artisan. Specialized culture medium may beobtained from commercial sources, or factors necessary or desirable forenhancing the proliferation may be added to standard culture medium.Additional factors and agents may also be added to culture medium, forexample, to induce expression of inducible elements in said cells or toinhibit growth of cells which are sensitive to particular agents.

By way of non-limiting example, DNA methylation inhibitors and histonedeacelyation inhibitors are two classes of agents that may be used inthe methods of the invention; exemplary agents include 5-aza-cytidine,TSA and valproic acid. As described herein, DNA methylation inhibitorsare also of use to identify cells that have been reprogrammed,regardless of whether a DNA methylation inhibitor contributes to thereprogramming. Thus in some embodiments of the invention thereprogramming agent is not a DNA methylation inhibitor, e.g., it has nodetectable effect on DNA methylation or reduces DNA methylation by lessthan 1%. In some embodiments the reprogramming agent reduces DNAmethylation by less than 5% and/or inhibits DNMT1, 3a, and/or 3bactivity by less than 1% or less than 5%.

In certain embodiments of the invention the reprogramming agent orfactor is exogenously introduced to the cell. “Exogenously introduced”is used consistently with its meaning in the art to refer to apolynucleotide (or other substance including but not limited to a smallmolecule or protein) which has been introduced into a cell or anancestor of the cell from outside the cell (typically by a process thatinvolves the hand of man) and/or is either not found in nature in cellsof that type or is found in a different sequence, context and/or indifferent amounts.

In some embodiments, reprogramming agents are introduced into cellsusing viral transduction, e.g., retroviral or lentiviral transduction.In particular embodiments the vector used is not subject tomethylation-induced silencing. In some embodiments the vector is anon-replicating vector, and in some embodiments the vector is anon-integrating vector. In particular embodiments the vector is anintegrating vector which is able to be excised from the cell's genome,e.g., able to be excised such that the cell's genome after excision issubstantially similar or identical to the genome of the cell prior tointegration of the vector. In some embodiments, reprogramming agents areintroduced into cells using protein transduction or transienttransfection of a nucleic acid construct that encodes a proteineffective either by itself or in combination with other reprogrammingagent(s) to reprogram the cells. Optionally cells are subjected to anelectric field and/or contacted with an agent that enhances cellpermeability to increase uptake of the reprogramming agent. In someembodiments, at least one of Oct4, Sox2, Klf4, Nanog, Lin28 and c-Mycmay be exogenously introduced into somatic cells using such methods. Inone embodiment Oct4, Sox2 and Klf4 are introduced into the cell(s),while in another embodiment Oct4, Sox2, Klf4 and c-Myc are introducedinto the cells(s). In another embodiment Oct4, Sox2, Nanog and Lin28 areintroduced into the cell(s).

Genes that affect the pluripotent state of a cell and thus are candidatereprogramming agents include pluripotency genes, genes involved inchromatin remodeling, and genes that are important for maintainingpluripotency, such as LIF, BMP, and PD098059 (Cell, 115: 281-292 (2003);Philos Trans R Soc Lond B Biol Sci. 2003 Aug. 29; 358(1436):1397-402).Thomson et al. used Oct4, Sox2, Nanog, and Lin28 using a lentiviralsystem to reprogram adult human cells (Thomson et al., Science 5854:1224-1225 (Nov. 23, 2007)). Other genes that can affect whether or not acell is pluripotent include certain oncogenes, such as c-myc. Othergenes include telomerase, e.g., the gene encoding the catalytic subunitof telomerase. Yet other genes include Sox1, Sox2, Sox3, Sox 15, Sox18,FoxD3, Stat3, N-Myc, L-Myc, Klf1, Klf2, Klf4 and Klf5. Other genes ofinterest include those encoding microRNA precursors that have beenassociated with multipotency or pluripotency and/or that is naturallyexpressed in multipotent or pluripotent cells. Optionally the gene isdownregulated as the cells differentiate and/or is not expressed inadult somatic cells. Other polynucleotides of interest include thoseencoding RNAi agents such as shRNAs targeted to a gene that is a targetof an endogenous microRNA that is naturally expressed in multipotent orpluripotent cells.

In addition, additional factors may be overexpressed or exogenouslyexpressed in the somatic cell to facilitate reprogramming. For example,factors which assist in inducing the cell to assume a lessdifferentiated state may be expressed in the cell. As described herein,C/EBPα has been shown to assist in the reprogramming of mature B cells.Other members of the C/EBPα family, such as human homologs of C/EBPα maybe similarly useful.

It will be understood that throughout the embodiments of the invention,encoded polypeptides may be exogenously introduced into a cell insteadof or in addition to exogenous introduction of a polynucleotide encodingsaid polypeptide unless otherwise indicated or implied from context. Inaddition, it will be understood that reference to a “gene” herein isintended to encompass the coding sequence of the gene with or withoutthe endogenous regulatory elements of the gene and with or withoutintronic sequence elements unless otherwise indicated or implied fromcontext.

Expression of an exogenously introduced polynucleotide may be carriedout in several ways. In one embodiment, the exogenously introducedpolynucleotide may be expressed from a chromosomal locus different fromthe endogenous chromosomal locus of the polynucleotide. Such chromosomallocus may be a locus with open chromatin structure, and contain gene(s)dispensible for a somatic cell. In other words, the desirablechromosomal locus contains gene(s) whose disruption will not cause cellsto die. Exemplary chromosomal loci include, for example, the mouse ROSA26 locus and type II collagen (Col2a1) locus (See Zambrowicz et al.,1997). The exogenously introduced polynucleotide may be expressed froman inducible promoter such that its expression can be regulated asdesired.

In an alternative embodiment, the exogenously introduced polynucleotidemay be transiently transfected into cells, either individually or aspart of a cDNA expression library. In one embodiment the cDNA expressionlibrary can be prepared from pluripotent cells, including but notlimited to embryonic stem cells, oocytes, blastomeres, inner cell masscells, embryonic germ cells, embryoid body (embryonic) cells,morula-derived cells, teratoma (teratocarcinoma) cells, and multipotentpartially differentiated embryonic stem cells taken from later in theembryonic development process. Candidate reprogramming agents may beidentified from such libraries.

The cDNA library is prepared by conventional techniques. Briefly, mRNAis isolated from an organism of interest. An RNA-directed DNA polymeraseis employed for first strand synthesis using the mRNA as template.Second strand synthesis is carried out using a DNA-directed DNApolymerase which results in the cDNA product. Following conventionalprocessing to facilitate cloning of the cDNA, the cDNA is inserted intoan expression vector such that the cDNA is operably linked to at leastone regulatory sequence. The choice of expression vectors for use inconnection with the cDNA library is not limited to a particular vector.Any expression vector suitable for use in mouse cells is appropriate. Inone embodiment, the promoter which drives expression from the cDNAexpression construct is an inducible promoter. The term regulatorysequence includes promoters, enhancers and other expression controlelements. Exemplary regulatory sequences are described in Goeddel; GeneExpression Technology: Methods in Enzymology, Academic Press, San Diego,Calif. (1990). For instance, any of a wide variety of expression controlsequences that control the expression of a DNA sequence when operativelylinked to it may be used in these vectors to express cDNAs. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, the lac system, the trp system, the TAC or TRC system,T7 promoter whose expression is directed by T7 RNA polymerase, the majoroperator and promoter regions of phage lambda, the control regions forfd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, thepromoters of the yeast α-mating factors, the polyhedron promoter of thebaculovirus system and other sequences known to control the expressionof genes of prokaryotic or eukaryotic cells or their viruses, andvarious combinations thereof. It should be understood that the design ofthe expression vector may depend on such factors as the choice of thehost cell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other protein encoded by thevector, such as antibiotic markers, should also be considered.

The exogenously introduced polynucleotide may be expressed from aninducible promoter. The term “inducible promoter”, as used herein,refers to a promoter that, in the absence of an inducer (such as achemical and/or biological agent), does not direct expression, ordirects low levels of expression of an operably linked gene (includingcDNA), and, in response to an inducer, its ability to direct expressionis enhanced. Exemplary inducible promoters include, for example,promoters that respond to heavy metals (CRC Boca Raton, Fla. (1991),167-220; Brinster et al. Nature (1982), 296, 39-42), to thermal shocks,to hormones (Lee at al. P.N.A.S. USA (1988), 85, 1204-1208; (1981), 294,228-232; Klock et al. Nature (1987), 329, 734-736; Israel and Kaufman,Nucleic Acids Res. (1989), 17, 2589-2604), promoters that respond tochemical agents, such as glucose, lactose, galactose or antibiotic(e.g., tetracycline or doxycycline).

A tetracycline-inducible promoter is an example of an inducible promoterthat responds to an antibiotic. See Gossen at al., 2003. Thetetracycline-inducible promoter comprises a minimal promoter linkedoperably to one or more tetracycline operator(s). The presence oftetracycline or one of its analogues leads to the binding of atranscription activator to the tetracycline operator sequences, whichactivates the minimal promoter and hence the transcription of theassociated cDNA. Tetracycline analogue includes any compound thatdisplays structural homologies with tetracycline and is capable ofactivating a tetracycline-inducible promoter. Exemplary tetracyclineanalogues includes, for example, doxycycline, chlorotetracycline andanhydrotetracycline. Also of use are tetracycline-repressible promoters.

The aforementioned methods may be used to express any of the exogenouslyintroduced polynucleotides described herein in a somatic cell. Forexample, they may be used to express a polynucleotide that encodes anRNAi agent targeted to an endogenous DNA methyltransferase or may beused to express a site-specific recombinase.

Applicant discovered that the exogenously introduced factors may bedispensable for maintenance of the pluripotent phenotype. For example,expression of exogenously introduced polynucleotides Oct4, Sox2 and Klf4are dispensable for maintenance of the pluripotent phenotype. Theinvention therefore comprises the recognition that reprogrammed somaticcells can be modified after being reprogrammed so as to render one ormore introduced factor(s), e.g., polynucleotides, nonfunctional whileretaining the ES-like phenotype of the cells.

In certain embodiments of the invention, rendering an introducedpolynucleotides nonfunctional reduces potential concerns associated withintroducing oncogenes into cells. Thus the invention comprisesintroducing one or more polynucleotides into a somatic cell, whereinsaid one or more polynucleotides at least in part reprogram the cell toan ES-like state, identifying a cell that has been reprogrammed to anES-like state, and functionally inactivating one or more of theintroduced polynucleotides. The cells may be maintained in culture for asuitable time period before inactivating the introducedpolynucleotide(s). In one embodiment the time period may be selected tobe sufficient for the cells to begin displaying a marker orcharacteristic of pluripotency, to begin expressing an endogenouspluripotency gene, e.g., Oct-4 and/or Nanog, or to begin expressing adownstream target of an endogenous pluripotency gene. In certainembodiments the exogenously introduced polynucleotide is regulated by aninducible regulatory element and functional inactivation is achieved byremoval of the inducer of said element.

Functional inactivation is also intended to encompass removal orexcision of the introduced polynucleotide. In certain embodiments atleast a portion of the one or more introduced polynucleotides is flankedby sites for a site-specific recombinase. The introduced polynucleotidecan be functionally inactivated by expressing the recombinase in thecell or introducing the recombinase into the cell. The resultingreprogrammed somatic cell may lack any exogenously introduced codingsequences and/or regulatory elements. The cell may be identical to anon-engineered somatic cell except that it contains one or more sitesthat remain following recombination.

Markers of Pluripotency

Somatic cells which have been treated with one or more reprogrammingagents are maintained in culture for a period of time sufficient tobegin reprogramming of the cell. Populations of treated cells may beanalyzed in a variety of ways to identify the occurrence ornon-occurrence of reprogramming. That is, a population of treated cellscan be further treated or analyzed to select for or enrich for cellswhich have begun the reprogramming process or to select against ordecrease cells which have not begun the reprogramming process.Populations of treated somatic cells can be assessed to identify cellswhich do or do not display one or more markers or characteristics ofreprogrammed, e.g., pluripotent, cells. For example, said cellpopulations can be assessed to identify phenotypic, functional orgenetic markers of reprogramming, including expression of one or morepluripotency genes and expression of one or more genes whose expressionis activated directly or indirectly as a result of expression of thepluripotency gene. By way of non-limiting example, a population of cellscan be assessed to identify expression of alkaline phosphatase,expression of SSEA1, expression of SSEA3, expression of SSEA4,expression of TRAF-60, expression of Nanog, expression of Oct4,expression of Fxb15, morphology characteristic of an ES cell or an EScell colony, ability to participate in formation of chimeras thatsurvive to term, ability to differentiate into cells havingcharacteristics of endoderm, mesoderm and ectoderm when injected intoSCID mice, presence of two active X chromosomes, resistance to DNAmethylation, and combinations thereof. A population of cells can also beassessed to identify the absence of any of the markers of reprogrammingto identify cells which have not undergone reprogramming.

The term “pluripotency gene”, as used herein, refers to a gene that isassociated with pluripotency. The expression of a pluripotency gene istypically restricted to pluripotent cells, e.g., pluripotent stem cells,and is crucial for the functional identity of pluripotent cells. It willbe appreciated that the protein encoded by a pluripotency gene may bepresent as a maternal factor in the oocyte, and the gene may beexpressed by at least some cells of the embryo, e.g., throughout atleast a portion of the preimplantation period and/or in germ cellprecursors of the adult.

In some embodiments the pluripotency gene is one whose averageexpression level in ES cells of a mammal is at least 5, 10, 20, 50, or100-fold greater than its average per cell expression level in somaticcell types present in the body of an adult mammal of that type (e.g.,mouse, human, farm animal). In some embodiments the pluripotency gene isone whose average expression level in ES cells is at least 5, 10, 20,50, or 100-fold greater than its average expression level in thoseterminally differentiated cell types present in the body of an adultmammal (e.g., mouse, human, farm animal). In some embodiments thepluripotency gene is one that is essential to maintain the viability orpluripotent state of ES cells derived using conventional methods. Thusif the gene is knocked out or inhibited (i.e., eliminated or reduced),the ES cells die or, in some embodiments, differentiate. In someembodiments the pluripotency gene is characterized in that inhibitingits expression in an ES cell (resulting in, e.g., a reduction in theaverage steady state level of RNA transcript and/or protein encoded bythe gene by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, ormore) results in a cell that is viable but no longer pluripotent. Insome embodiments the pluripotency gene is characterized in that itsexpression in an ES cell decreases (resulting in, e.g., a reduction inthe average steady state level of RNA transcript and/or protein encodedby the gene by at least 50%, 60%, 70%, 80%, 90%, 95%, or more) when thecell differentiates into a terminally differentiated cell.

The transcription factor Oct-4 (also called Pou5fl, Oct-3, Oct3/4) is anexample of a pluripotency gene. Oct-4 has been shown to be required forestablishing and maintaining the undifferentiated phenotype of ES cellsand plays a major role in determining early events in embryogenesis andcellular differentiation (Nichols et al., 1998, Cell 95:379-391; Niwa etal., 2000, Nature Genet. 24:372-376). Oct-4 is down-regulated as stemcells differentiate into specialised cells.

Nanog is another example of a pluripotency gene. Nanog is a divergenthomeodomain protein that directs propagation of undifferentiated EScells. Nanog mRNA is present in pluripotent mouse and human cell lines,and absent from differentiated cells. In pre-implantation embryos, Nanogis restricted to founder cells from which ES cells can be derived.Endogenous Nanog acts in parallel with cytokine stimulation of Stat3 todrive ES cell self-renewal. Elevated Nanog expression from transgeneconstructs is sufficient for clonal expansion of ES cells, bypassingStat3 and maintaining Oct4 levels. (See Chambers et al., 2003, Cell 113:643-655; Mitsui et al., Cell. 2003, 113(5):631-42). Other exemplarypluripotency genes include Sox2 and Stella (see Imamura et al., BMCDevelopmental Biology 2006, 6:34, Bortvin et al. Development. 2003,130(8):1673-80; Saitou et al., Nature. 2002, 418 (6895):293-300).

In certain embodiments of the invention the endogenous pluripotency geneis co-expressed with a selectable marker. For example, the endogenouspluripotency gene can be linked to a polynucleotide (e.g., DNA) encodinga selectable marker in such a manner that the selectable marker and theendogenous pluripotency gene are co-expressed. As used hereinco-expression is intended to mean that expression of the selectablemarker substantially matches the expression of the endogenouspluripotency gene. In one embodiment, the differentiated somatic cellsof the present invention comprise a first endogenous pluripotency genelinked to DNA encoding a first selectable marker in such a manner thatthe expression of the first selectable marker substantially matches theexpression of the first endogenous pluripotency gene. The differentiatedsomatic cells may also be engineered to comprise any number ofendogenous pluripotency genes respectively linked to a distinctselectable marker. Thus, in another embodiment, the differentiatedsomatic cells of the present invention comprise two endogenouspluripotency genes, each of which is linked to DNA encoding a distinctselectable marker. In a further embodiment, the differentiated somaticcells of the present invention comprise three endogenous pluripotencygenes, each of which is linked to DNA encoding a distinct selectablemarker. The differentiated somatic cells may be further engineered tohave one or more pluripotency gene(s) expressed as a transgene under aninducible promoter.

In one embodiment, somatic cells used in the methods comprise only oneendogenous pluripotency gene linked to a first selectable marker, andthe selection step is carried out to select for the expression of thefirst selectable marker. In an alternative embodiment, the somatic cellsused in the methods comprise any number of endogenous pluripotencygenes, each of which is linked to a distinct selectable markerrespectively, and the selection step is carried out to select for atleast a subset of the selectable markers. For example, the selectionstep may be carried out to select for all the selectable markers linkedto the various endogenous pluripotency genes.

In one embodiment, somatic cells used in the method comprise aselectable marker linked to an endogenous pluripotency gene and anadditional pluripotency gene expressed as a transgene under an induciblepromoter. For these cells, the method of reprogramming may compriseinducing the expression of the pluripotency transgene and select for theexpression of the selectable marker. The method may further comprisecontacting the somatic cells with an agent that alters chromatinstructure.

For purposes of the present invention, it is not necessary that theexpression level of the endogenous pluripotency gene and the selectablemarker is the same or even similar. It is only necessary that the cellsin which an endogenous pluripotency gene is activated will also expressthe selectable marker at a level sufficient to confer a selectablephenotype on the reprogrammed cells. For example, when the selectablemarker is a marker that confers resistance to a lethal drug (a “drugresistance marker”), the cells are engineered in a way that allows cellsin which an endogeneous pluripotency gene is activated to also expressthe drug resistance marker at a sufficient level to confer onreprogrammed cells resistance to lethal drugs. Thus, reprogrammed cellswill survive and proliferate whereas non-reprogrammed cells will die.

In certain embodiments of the invention the selectable marker isoperably linked to expression control elements that regulatetranscription from the endogenous pluripotency gene. The DNA encoding aselectable marker may be inserted downstream from the end of the openreading frame (ORF) encoding the desired endogenous pluripotency gene,anywhere between the last nucleotide of the ORF and the first nucleotideof the polyadenylation site. An internal ribosome entry site (IRES) maybe placed in front of the DNA encoding the selectable marker.Alternatively, the DNA encoding a selectable marker may be insertedanywhere within the ORF of the desired endogenous pluripotency gene,downstream of the promoter, with a termination signal. An internalribosome entry site (IRES) may be placed in front of the DNA encodingthe selectable marker. In further embodiments the DNA encoding theselectable marker may be inserted anywhere within a gene whoseexpression is activated directly or indirectly as a result of expressionof the pluripotency gene. In some embodiments the DNA encoding aselectable marker is inserted into an intron. In some embodiments, theendogenous pluripotency gene into which the DNA has been insertedexpresses a functional pluripotency gene product while in otherembodiments it does not. The selectable marker may be inserted into onlyone allele, or both alleles, of the endogenous pluripotency gene. Incertain other embodiments an exogenous polynucleotide including aselectable marker operably linked to expression control elements thatregulate transcription from the endogenous pluripotency gene is insertedinto the cellular genome at a location external to the locus of anendogenous pluripotency gene such that conditions appropriate toactivate expression of the endogenous pluripotency gene also activateexpression of the exogenous polynucleotide.

A selectable marker, as used herein, is a marker that, when expressed,confers upon recipient cells a selectable phenotype, such as antibioticresistance, resistance to a cytotoxic agent, nutritional prototrophy, orexpression of a surface protein. Other proteins whose expression can bereadily detected such as a fluorescent or luminescent protein or anenzyme that acts on a substrate to produce a colored, fluorescent, orluminescent substance are also of use as selectable markers. Thepresence of a selectable marker linked to an endogenous pluripotencygene makes it possible to identify and select reprogrammed cells inwhich the endogenous pluripotency gene is expressed. A variety ofselectable marker genes can be used, such as neomycin resistance gene(neo), puromycin resistance gene (puro), guanine phosphoribosyltransferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase(ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene(hyg), multidrug resistance gene (mdr), thymidine kinase (TK),hypoxanthine-guanine phosphoribosyltransferase (HPRT), and hisD gene.Other markers include green fluorescent protein (GFP) blue, sapphire,yellow, red, orange, and cyan fluorescent proteins and variants of anyof these. Luminescent proteins such as luciferase (e.g., firefly orRenilla luciferase) are also of use. Systems based on enzyme reporterssuch as beta-galactosidase, alkaline phosphatase, chloramphenicolacetyltransferase, etc., are also of use. In some embodiments the markeris a secreted enzyme. As will be evident to one of skill in the art, theterm “selectable marker” as used herein can refer to a gene or to anexpression product of the gene, e.g., an encoded protein.

In some embodiments the selectable marker confers a proliferation and/orsurvival advantage on cells that express it relative to cells that donot express it or that express it at significantly lower levels. Suchproliferation and/or survival advantage typically occurs when the cellsare maintained under certain conditions, i.e., “selective conditions”.To ensure an effective selection, a population of cells can bemaintained for a under conditions and for a sufficient period of timesuch that cells that do not express the marker do not proliferate and/ordo not survive and are eliminated from the population or their number isreduced to only a very small fraction of the population. The process ofselecting cells that express a marker that confers a proliferationand/or survival advantage by maintaining a population of cells underselective conditions so as to largely or completely eliminate cells thatdo not express the marker is referred to herein as “positive selection”,and the marker is said to be “useful for positive selection”. Markersuseful for positive selection are of particular interest in embodimentsof the invention in which an endogenous pluripotency gene is linked to aselectable marker.

Negative selection and markers useful for negative selection are also ofinterest in certain of the methods described herein. Expression of suchmarkers confers a proliferation and/or survival disadvantage on cellsthat express the marker relative to cells that do not express the markeror express it at significantly lower levels (or, considered another way,cells that do not express the marker have a proliferation and/orsurvival advantage relative to cells that express the marker). Cellsthat express the marker can therefore be largely or completelyeliminated from a population of cells when maintained in selectiveconditions for a sufficient period of time.

Certain markers of interest herein are useful for positive and negativeselection depending on the particular selective conditions employed.Thus under certain sets of conditions cells that express the marker havea proliferation and/or survival advantage relative to cells that do notexpress the marker while under other sets of conditions cells thatexpress the marker have a proliferation and/or survival disadvantagerelative to cells that do not express the marker. Two examples of suchmarkers that are suitable for use in the invention are hypoxanthinephosphoribosyl transferase (HPRT), an enzyme that catalyzes certainreactions in which purine-type compounds are synthesized and/orinterconverted, and thymidine kinase (TK), which catalyzes certainreactions in which pyrimidine-type compounds are synthesized and/orinterconverted. Under typical culture conditions DNA synthesis inmammalian cells proceeds through a main (de novo) pathway in whichglutamine and aspartate are used as initial substrates for a series ofreactions leading to synthesis of purine-type (e.g., dATP and dGTP) andpyrimidine-type (e.g., dCTP and dTTP) nucleotides. When the de novopathway is blocked, mammalian cells must utilize alternative pathways tosynthesize the needed nucleotides. The purine salvage pathway involvesconverting hypoxanthine to IMP, a reaction catalyzed by HPRT. The secondpathway converts thymidine to dTMP, a reaction catalyzed by TK. Thuscells lacking HPRT expression (e.g., cells lacking a functional copy ofthe HPRT gene) or lacking TK expression (e.g., cells lacking afunctional copy of the TK gene) can grow in standard culture medium butdie in HAT medium, which contains aminopterin, hypoxanthine, andthymidine. In cells lacking HPRT endogenous expression, HPRT can be usedas selectable marker whose expression may be selected for in HAT medium.Similarly, in cells lacking endogenous TK expression, TK can be used asa selectable marker whose expression may be selected for in HAT medium.

In addition to the ability to select for cells that express HPRT or TK,it is also possible to select for cells that lack expression offunctional HPRT and/or TK, e.g., cells that do not express one or bothof these enzymes. HPRT converts certain otherwise non-toxic compoundsincluding a variety of purine analogs such as 8-azaguanine (8-AZ) and6-thioguanine (6-TG) into cytotoxic compounds. TK converts certainpyrimidine analogs such as 5-bromodeoxyuridine andtrifluoro-methyl-thymidine into cytotoxic compounds. The cytotoxiccompounds may have deleterious effects on cells e.g., by inhibitingenzymes involved in nucleic acid synthesis and/or becoming incorporatedinto DNA, leading to mismatches and mutations. Thus in culture mediumcontaining 8-AZ, 6-TG, etc., cells that express HPRT are at a growthdisadvantage relative to cells that do not express HPRT or that expressit at lower levels insufficient to fully support nucleic acid synthesis,It is therefore possible to use these selective conditions to select forcells that lack HPRT activity. Similarly, in medium containingbromodeoxyuridine or trifluoro-methyl-thymidine, cells that express TKare at a growth disadvantage relative to cells that lack TK expressionor express a lower and insufficient level of TK. It is thereforepossible to use these selective conditions to select for cells that lackTK activity.

In some embodiments of the invention, a population of differentiatedsomatic cells which have been treated with one or more reprogrammingagents or factors and then maintained for a suitable period of time areassayed to identify cells which display a marker of pluripotency.

As described herein, differentiated somatic cells for use in theinvention may the engineered differentiated somatic cells can beobtained from a transgenic mouse comprising such engineered somaticcells. Such transgenic mouse can be produced using standard techniquesknown in the art. For example, Bronson et al. describe a technique forinserting a single copy of a transgene into a chosen chromosomal site.See Bronson et al., 1996. Briefly, a vector containing the desiredintegration construct (for example, a construct containing a selectablemarker linked to a pluripotency gene) is introduced into ES cells bystandard techniques known in the art. The resulting ES cells arescreened for the desired integration event, in which the knock-in vectoris integrated into the desired endogenous pluripotency gene locus suchthat the selectable marker is integrated into the genomic locus of thepluripotency gene and is under the control of the pluripotency genepromoter. The desired ES cell is then used to produce a transgenic mousein which all cell types contain the correct integration event. Desiredtypes of cells may be selectively obtained from the transgenic mouse andmaintained in vitro. In one embodiment, two or more transgenic mice maybe created, each carrying a distinct integration construct. These micemay then be bred to generate mice that carry multiple desiredintegration constructs. For example, one type of transgenic mouse may becreated to carry an endogenous pluripotency gene linked to a selectablemarker, while a second type of transgenic mouse may be created to carrya pluripotency gene expressed as a transgene under an induciblepromoter. These two types of mice may then be bred to generatetransgenic mice that have both a selectable marker linked to anendogenous pluripotency gene and an additional pluripotency geneexpressed as a transgene under an inducible promoter. These twopluripotency genes may or may not be the same. Many variables arecontemplated: the identity of the endogenous pluripotency gene linked tomarker, the identity of the pluripotency gene expressed as a transgene,and the number of the endogenous pluripotency gene linked to aselectable marker, and the number of pluripotency gene expressed as atransgene. The present invention encompasses all possible combinationsof these variables. In other embodiments one of the mice carries anendogenous pluripotency gene linked to a selectable marker and one ofthe mice carries a DNA that encodes an RNAi agent targeted to a DNMTgene (thereby capable of inhibiting the expression of the DNMT gene) asdiscussed further below.

Alternatively, engineered differentiated somatic cells of the presentinvention may be produced by direct introduction of the desiredconstruct into somatic cells. A DNA construct may be introduced intocells by any standard technique known in the art, such as viraltransfection (e.g., using an adenoviral system) or liposome-mediatedtransfection. Any means known in the art to generate somatic cells withtargeted integration can be used to produce somatic cells of theinvention, e.g., cells in which a selectable marker is operably linkedto an endogenous pluripotency gene or cells in which an endogenous geneis rendered conditional by introducing a conditional promoter or sitesfor a site-specific recombinase into or near the gene.

In mammalian cells, homologous recombination occurs at much lowerfrequency compared to non-homologous recombination. To facilitate theselection of homologous recombination events over the non-homologousrecombination events, at least two enrichment methods have beendeveloped: the positive-negative selection (PNS) method and the“promoterless” selection method (Sedivy and Dutriaux, 1999). Briefly,PNS, the first method, is in genetic terms a negative selection: itselects against recombination at the incorrect (non-homologous) loci byrelying on the use of a negatively selectable gene that is placed on theflanks of a targeting vector. On the other hand, the second method, the“promoterless” selection, is a positive selection in genetic terms: itselects for recombination at the correct (homologous) locus by relyingon the use of a positively selectable gene whose expression is madeconditional on recombination at the homologous target site. Thedisclosure of Sedivy and Dutriaux is incorporated herein.

As described herein, differentiated somatic cells which have beencontacted with at least one reprogramming agent are assessed todistinguish cells which have been reprogrammed to multipotency orpluripotency from cells which have not. This may be done bydistinguishing cells which demonstrate one or more pluripotencycharacteristics or display one or more markers of pluripotency fromcells which do not.

The term “pluripotency characteristics”, as used herein, refers to manycharacteristics associated with pluripotency, including, for example,the ability to differentiate into all types of cells and an expressionpattern distinct for a pluripotent cell, including expression ofpluripotency genes, expression of other ES cell markers, and on a globallevel, a distinct expression profile known as “stem cell molecularsignature” or “stemness.”

Thus, to assess reprogrammed somatic cells for pluripotencycharacteristics, one may analyze such cells for different growthcharacteristics and ES cell-like morphology. Cells may be injectedsubcutaneously into immunocompromised SCID mice to induce teratomas (astandard assay for ES cells). ES-like cells can be differentiated intoembryoid bodies (another ES specific feature). Moreover, ES-like cellscan be differentiated in vitro by adding certain growth factors known todrive differentiation into specific cell types. Self-renewing capacity,marked by induction of telomerase activity, is another plutipotencycharacteristic that can be monitored. One may carry out functionalassays of the reprogrammed somatic cells by introducing them intoblastocysts and determine whether the cells are capable of giving riseto all cell types. See Hogan et al., 2003. If the reprogrammed cells arecapable of forming a few cell types of the body, they are multipotent;if the reprogrammed cells are capable of forming all cell types of thebody including germ cells, they are pluripotent.

One may also examine the expression of an individual pluripotency genein the reprogrammed somatic cells to assess their pluripotencycharacteristics. Additionally, one may assess the expression of other EScell markers. Stage-specific embryonic 1 5 antigens-1, -3, and -4(SSEA-1, SSEA-3, SSEA-4) are glycoproteins specifically expressed inearly embryonic development and are markers for ES cells (Solter andKnowles, 1978, Proc. Natl. Acad. Sci. USA 75:5565-5569; Kannagi et al.,1983, EMBO J 2:2355-2361). Elevated expression of the enzyme AlkalinePhosphatase (AP) is another marker associated with undifferentiatedembryonic stem cells (Wobus et al., 1984, Exp. Cell 152:212-219; Peaseet al., 1990, Dev. Biol. 141:322-352). Other stem/progenitor cellsmarkers include the intermediate neurofilament nestin (Lendahl et al.,1990, Cell 60:585-595; Dah-Istrand et al., 1992, J. Cell Sci.103:589-597), the membrane glycoprotein prominin/AC133 (Weigmann et al.,1997, Proc. Natl. Acad. USA 94:12425-12430; Corbeil et al., 1998, Blood91:2625-22626), the transcription factor Tcf-4 (Korinek et al, 1998,Nat. Genet. 19: 379-383; Lee et al., 1999, J. Biol. Chem. 274.1 566-1572), and the transcription factor Cdx1 (Duprey et al., 1988, Genes Dev.2:1647-1654; Subramania'n et al., 1998, Differentiation 64:11-18).Additional ES cell markers are described in Ginis, I., et al., Dev.Biol., 269: 369-380, 2004. For example, REX-1, TEAT, UTF-1, TRF-1,TRF-2, connexin43, connexin45, FGFR-4, ABCG-2, and Glut-1 are of use.

One may additionally conduct expression profiling analyses of thereprogrammed somatic cells to assess their pluripotency characteristics.Pluripotent cells, such as embryonic stem cells, and multipotent cells,such as adult stem cells, are known to have a distinct pattern of globalgene expression profile. This distinct pattern is termed “stem cellmolecular signature”, or “stemness”. See, for example, Ramalho-Santos etal., Science 298: 597-600 (2002); Ivanova et al., Science 298: 601-604.One may assess the epigenetic state of cellular DNA. One may assess theresistance of the cells to global DNA demethylation. One may assess thedevelopmental potential of the cells. In some embodiments, cells thatare able to form teratomas containing cells having characteristics ofendoderm, mesoderm, and ectoderm when injected into SCID mice and/orpossess ability to participate (following injection into murineblastocysts) in formation of chimeras that survive to term areconsidered pluripotent.

Engineered Somatic Cells and Transgenic Mice Comprising Such Cells

The present invention further provides engineered somatic cells in whichDNA methylation can be regulated. “DNA methylation” is used hereinconsistently with it use in the art to refer to the modification ofeukaryotic DNA by attachment of a methyl group to a cytosine. As knownin the art, cytosine methylation of DNA plays important roles inepigenetic gene regulation and the maintenance of genomic integrity.Mammalian cells possess several different DNA methyltransferases thatare responsible for transfer of a methyl group to cytosine present inDNA (Goll, G, and Bestor, T., Annu Rev. Biochemistry, 74: 481-514,2005). As least three genes are involved in establishing and maintaininggenomic methylation in mammalian cells, i.e., those encoding the de novomethyltransferases DNMT3a and DNMT3b and the maintenance enzyme DNMT1which methylates hemimethylated DNA but also exhibits the ability tomethylate unmethylated DNA. Mutational analysis in mice has demonstratedthat these three genes are essential, with lethality occurring soonafter gastrulation in Dnmt1-null embryos and at later time points in thecase of embryos lacking functional Dnmt3a or Dnmt3b genes (Li, 1992;Okano, at al., Cell, 99(3):247-57, 1999). Aberrant regulation of anumber of genes has been observed in these embryos. The data areconsistent with a requirement for DNA methylation for thetranscriptional silencing that occurs in many cell types duringmammalian development and is likely necessary for the proper celldifferentiation.

The invention provides cells in which expression of an endogenous DNAmethyltransferase (DNMT) gene such as Dnmt1, Dnmt3a, or Dnmt3b can beregulated and/or in which expression of an endogenous Dnmt gene isaltered relative to nonengineered somatic cells. In certain embodimentsthe somatic cells contain an exogenously introduced gene that encodes anRNA that interferes with expression of an endogenous DNAmethyltransferase (DNMT) gene such as Dnmt1, Dnmt3a, or Dnmt3b. In someembodiments the RNA interferes with expression of an endogenous DNAmethyltransferase gene by RNA interference (RNAi). “RNAi” is used hereinconsistently with its meaning in the art to refer to a phenomenonwhereby double-stranded RNA (dsRNA) triggers the sequence-specificdegradation or translational repression of a corresponding mRNA havingcomplementarity to one strand of the dsRNA. It will be appreciated thatthe complementarity between the strand of the dsRNA and the mRNA neednot be 100% but need only be sufficient to mediate inhibition of geneexpression (also referred to as “silencing” or “knockdown”). Forexample, the degree of complementarity is such that the strand caneither (i) guide cleavage of the mRNA in a protein complex called theRNA-induced silencing complex (RISC); or (ii) cause translationalrepression of the mRNA. In certain embodiments the double-strandedportion of the RNA is less than about 30 nucleotides in length, e.g.,between 17 and 29 nucleotides in length. In mammalian cells, RNAi may beachieved by introducing an appropriate double-stranded nucleic acid intothe cells or expressing a nucleic acid in cells that is then processedintracellularly to yield dsRNA therein.

For purposes of the present invention an at least partly double-strandedRNA that is capable of triggering sequence-specific inhibition of geneexpression, optionally after undergoing intracellular processing, isreferred to as an “RNAi agent”. Exemplary nucleic acids capable ofmediating RNAi are a short hairpin RNA (shRNA), a short interfering RNA(siRNA), and a microRNA precursor. These terms are well known and areused herein consistently with their meaning in the art. siRNAs typicallycomprise two separate nucleic acid strands that are hybridized to eachother to form a duplex. They can be synthesized in vitro, e.g., usingstandard nucleic acid synthesis techniques. They can comprise a widevariety of modified nucleosides, nucleoside analogs and can comprisechemically or biologically modified bases, modified backbones, etc. Anymodification recognized in the art as being useful for RNAi can be used.Some modifications result in increased stability, cell uptake, potency,etc. In certain embodiments the siRNA comprises a duplex about 19nucleotides in length and one or two 3′ overhangs of 1-5 nucleotides inlength, which may be composed of deoxyribonucleotides. shRNA comprise asingle nucleic acid strand that contains two complementary portionsseparated by a predominantly non-selfcomplementary region. Thecomplementary portions hybridize to form a duplex structure and thenon-selfcomplementary region forms a loop connecting the 3′ end of onestrand of the duplex and the 5′ end of the other strand. shRNAs undergointracellular processing to generate siRNAs.

MicroRNAs (miRNAs) are small, non-coding, single-stranded RNAs of about21-25 nucleotides (in mammalian systems) that inhibit gene expression ina sequence-specific manner. They are generated intracellularly fromprecursors having a characteristic secondary structure comprised of ashort hairpin (about 70 nucleotides in length) containing a duplex thatoften includes one or more regions of imperfect complementarity.Naturally occurring miRNAs are only partially complementary to theirtarget mRNA and typically act via translational repression. As usedherein the term “shRNA” encompasses RNAi agents modelled on endogenousmicroRNA precursors. In some embodiments, a sequence encoding the stemportion of a stem-loop structure or encoding a complete stem-loop can beinserted into a nucleic acid comprising at least a portion of anendogenous microRNA primary transcript, e.g., in place of the sequencethat encodes the endogenous microRNA or minimum (˜70 nucleotide)microRNA hairpin.

One of skill in the art will be able to identify an appropriate RNAiagent to inhibit expression of a gene. Such an RNAi agent is referred toas being “targeted to” the gene and the encoded mRNA. The RNAi agent mayinhibit expression sufficiently to reduce the average steady state levelof the RNA transcribed from the gene (e.g., mRNA) or its encoded proteinby, e.g., by at least 50%, 60%, 70%, 80%, 90%, 95%, or more). The RNAiagent may contain a sequence between 17-29 nucleotides long, e.g., 19-23nucleotides long that is 100% complementary to the mRNA or contains upto 1, 2, 3, 4, or 5 nucleotides, or up to about 10-30% nucleotides, thatdo not participate in Watson-Crick base pairs when aligned with the mRNAto achieve the maximum number of complementary base pairs. The RNAiagent may contain a duplex between 17-29 nucleotides long in which allnucleotides participate in Watson-Crick base pairs or in which up toabout 10-30% of the nucleotides do not participate in a Watson-Crickbase pair. One of skill in the art will be aware of which sequencecharacteristics are often associated with superior siRNA functionalityand algorithms and rules by which such siRNAs can be designed (see,e.g., Jagla, B., et al, RNA, 11(6):864-72, 2005). The methods of theinvention can employ siRNAs having such characteristics, although therange of useful sequences is not limited to those that satisfy theserules. In some embodiments the sequence of either or both strands of theRNAi agent is/are chosen to avoid silencing non-target genes, e.g., thestrand(s) may have less than 70%, 80%, or 90% complementarity to anymRNA other than the target mRNA. In some embodiments multiple differentsequences are used. The tables below list the Gene IDs of the human andmouse genes encoding DNMT1, 3a, and 3b and antisense sequences ofexemplary siRNAs for silencing these genes. Similar information isincluded also for HPRT. One of skill in the art can readily find theGene ID, accession numbers, and sequence information for any gene ofinterest in publicly available databases. One of skill in the art canreadily design siRNAs and shRNAs to silence these genes or others. Itwill be appreciated that the sequences may be varied and/or extended byincorporating additional nucleotides at either or both ends.Furthermore, if multiple isoforms exist, one can design siRNAs or shRNAstargeted against a region present in all of the isoforms expressed in agiven cell type or organism of interest.

TABLE A siRNA sequences targeting Human genes Gene Gene IDsiRNA sequences Dnmt1 1786 GGAAGAAGAGUUACUAUAA  (SEQ. ID. NO: 11)GAGCGGAGGUGUCCCAAUA  (SEQ. ID. NO: 12) GGACGACCCUGACCUCAAA (SEQ. ID. NO: 13) GAACGGUGCUCAUGCUUAC  (SEQ. ID. NO: 14)UUUCUCCCUCAGACACUC  (SEQ ID NO: 15) Dnmt3a 1788 GCACAAGGGUACCUACGGG (SEQ. ID. NO: 16) CAAGAGAGCGGCUGGUGUA  (SEQ. ID. NO: 17)GCACUGAAAUGGAAAGGGU  (SEQ. ID. NO: 18) GAACUGCUUUCUGGAGUGU (SEQ. ID. NO: 19) Dnmt3b 1789 GAAAGUACGUCGCUUCUGA  (SEQ. ID. NO: 20)ACAAAUGGCUUCAGAUGUU  (SEQ. ID. NO: 21) GCUCUUACCUUACCAUCGA (SEQ. ID. NO: 22) UUUACCACCUGCUGAAUUA  (SEQ. ID. NO: 23) Hprt 3251CCAGUUUCACUAAUGACACAA  (SEQ ID NO: 24)

TABLE B siRNA Targeting Mouse genes Gene Gene ID siRNA sequences Dnmt113433 GGAAAGAGAUGGCUUAACA  (SEQ. ID. NO: 25) GCUGGGAGAUGGCGUCAUA (SEQ. ID. NO: 26) GAUAAGAAACGCAGAGUUG  (SEQ. ID. NO: 27)GGUAGAGAGUUACGACGAA  (SEQ. ID. NO: 28) Dnmt3a 13435 CGCGAUUUCUUGAGUCUAA (SEQ. ID. NO: 29) CGAAUUGUGUCUUGGUGGA  (SEQ. ID. NO: 30)AAACAUCGAGGACAUUUGU  (SEQ. ID. NO: 31) CAAGGGACUUUAUGAGGGU (SEQ. ID. NO: 32) Dnmt3b 13436 GCAAUGAUCUCUCUAACGU  (SEQ. ID. NO: 33)GGAAUGCGCUGGGUACAGU  (SEQ. ID. NO: 34) UAAUCUGGCUACCUUCAAU (SEQ. ID. NO: 35) GCAAAGGUUUAUAUGAGGG  (SEQ. ID. NO: 36) Plort 15452CCAGUUUCACUAAUGACACAA  (SEQ ID NO: 37)

To express an RNAi agent in somatic cells, a nucleic acid constructcomprising a sequence that encodes the RNAi agent, operably linked tosuitable expression control elements, e.g., a promoter, can beintroduced into the cells as known in the art. For purposes of thepresent invention a nucleic acid construct that comprises a sequencethat encodes an RNA or polypeptide of interest, the sequence beingoperably linked to expression control elements such as a promoter thatdirect transcription in a cell of interest, is referred to as an“expression cassette”. The promoter can be an RNA polymerase I, II, orIII promoter functional in mammalian cells. In certain embodiments thepromoter is one that is functional when introduced into somatic cells.In certain embodiments expression of the RNAi agent is conditional. Insome embodiments expression is regulated by placing the sequence thatencodes the RNAi agent under control of a regulatable (e.g., inducibleor repressible) promoter.

In some embodiments regulation of expression of a DNA methyltransferaseis dependent on a site-specific recombinase. Site-specific recombinasesand methods of use thereof for achieving controlled and reversibleexpression of genes are known in the art. Such recombinases are proteinsthat recognize specific nucleic acid sequences and mediate insertioninto or excision of sequences located between these sites. Recombinasesystems include the Cre-Lox and Flp-Frt systems, among others. In someembodiments at least a portion of the coding sequence for the RNAi agentis positioned between sites for the recombinase. Expression of therecombinase (e.g., Cre) in the cell or its exogenous introduction into acell causes excision of the portion of the coding sequence locatedbetween the sites, permanently turning off expression of the gene. Insome embodiments expression of a gene in a cell is prevented due topresence of a “stopper” sequence located between a promoter element andthe transcription start site or between different portions of a promoterelement (e.g., between a TATA box and a second portion of a promoterelement). The stopper sequence is flanked by sites for a recombinase,which sites are also located between the promoter and the transcriptionstart site or between different portions of a promoter element.Expression or introduction of the recombinase into the cell causesexcision of the stopper sequence, thereby bringing the promoter intooperable association with the transcription start site or reconstitutinga functional promoter, thereby allowing transcription to proceed. Insome embodiments, the cells comprise an expression cassette in whichexpression of the recombinase is under control of inducible expressioncontrol elements such as an inducible promoter. Expression of therecombinase is induced, e.g., by administering an appropriate inducingagent such as a small molecule (e.g., tetracycline or an analog thereof,a hormone such as estrogen or a glucocorticoid, a metal, etc.) to cellsor to an organism or by introducing an expression vector that encodesthe recombinase into the cell or organism. See, e.g., U.S. Pat. No.6,995,011 and Ventura, at al. (reference 13 of reference set 2). In oneembodiment the promoter is a U6 promoter, and a Lox-Stop-Lox sequence isinserted between the proximal sequence element (PSE) and the TATA box orbetween the TATA box and the transcription start site. In someembodiments the TATA box in a promoter (e.g., the U6 promoter) isreplaced by a bifunctional lox site (TATAlox) that retains the abilityto undergo Cre-mediated recombination and contains a functional TATA boxis used, so that after recombination the spacing between the PSE, TATA,and transcriptional start site is not altered (Ventura, et. Al, 2004).

In some embodiments the invention provides a cell that comprises a firstcopy of a Dnmt gene that is functional but can be rendered nonfunctionalby expressing in or introducing a first recombinase into the cell and asecond copy of the Dnmt gene that is nonfunctional but can be renderedfunctional by expressing in or introducing a second recombinase into thecell. The first copy of the gene or an essential portion thereof may,for example, be flanked by sites for the first recombinase so that whenthe first recombinase is present the gene or a portion thereof isexcised and the gene is rendered nonfunctional. The second copy of thegene may, for example, comprise a stopper sequence located between sitesfor the second recombinase. The stopper sequence prevents synthesis of afunctional DNMT protein. For example, the stopper may be present betweenthe promoter and the transcriptional start site and preventtranscription or it may result in an insertion into the DNMT proteinthat renders the protein non-functional. When the second recombinase ispresent the stopper sequence is excised, and a functional DNMT isproduced. In some embodiments, a gene is considered “nonfunctional” ifit is not detectably transcribed or, if transcribed, the level oftranscription is reduced by at least 100-fold. In some embodiments, a“nonfunctional” gene encodes a DNMT protein that lacks at least 90% ofits catalytic domain and/or at least 90% of its localization domain. Oneof skill in the art will be able to generate non-functional Dnmt1, 3a,and/or 3b genes. Genes can be tested to determine whether they encode afunctional protein using standard in vitro assays or by determiningwhether the gene is able to rescue the lethality of a Dnmt1, 3a, or 3bknockout. In some embodiments, a “nonfunctional gene” encodes a proteinwhose DNA methylating activity in vitro against a suitable substrate(e.g., hemimethylated DNA in the case of DNMT1) using a standard assayknown in the art is reduced by at least 95%, 98%, 99% or more. In someembodiments a non-functional gene is one that, when present as the solesource of DNMT protein in a somatic cell of interest such as a primarymammalian fibroblast, does not encode a protein capable of allowing thecell to survive for a period of 10 days in standard culture conditions.DNA methylation can be regulated in the cell as follows. First, DNAmethylation is inhibited by introduction or expression of the firstrecombinase, thereby eliminating expression of functional DNMT1. Thecells are maintained in culture. In the absence of a functional DNMT1,DNA demethylation occurs over time (either spontaneously or as a resultof active demethylation) and hemimethylated DNA is not remethylatedafter cell division. When it is desired to restore DNA methylation, thesecond recombinase is introduced or expressed in cells, causing removalof the stopper sequence and allowing production of functional DNMT1. Insome embodiments this approach is applied to render expression of DNMT1,3a, 3b, or any combination thereof conditional.

In some embodiments the recombinase is expressed transiently, e.g., itbecomes undetectable after about 1-2 days, 2-7 days, 1-2 weeks, etc.Transient expression can be achieved by transient transfection or byexpression from a regulatable promoter.

In some embodiments the recombinase is introduced from external sources.Optionally the recombinase in these embodiments comprises an amino acidsequence (also referred to as a “protein transduction domain”) thatenhances cellular uptake of polypeptides. Such uptake-enhancing aminoacid sequences are found, e.g., in HIV-1 TAT protein, the herpes simplexvirus 1 (HSV-1) DNA-binding protein VP22, the Drosophila Antennapedia(Antp) homeotic transcription factor, and others. Synthetic peptides,e.g., having a high basic amino acid content (Lys and Arg) are also ofuse. See U.S. Patent Pub. No. 20060148104 for additional usefulsequences. In some embodiments expression of the recombinase is achievedby infecting cells with a vector, e.g., a virus vector (e.g., alentivirus, adenovirus, or adeno-associated virus vector) containing anexpression cassette containing the sequence encoding the recombinaseoperably linked to a promoter. The vector may be one that results intransient expression of the recombinase, e.g., that does not stablyintegrate into the cell's genome or result in a stably inheritedepisome. In certain embodiments of the invention the engineered somaticcells contain a functional p53 pathway (see Harris, S., and Levine, A,Oncogene, 24: 2899-2908, 2005 for description of p53 pathways). Suchcells contain a functional p53 gene and are able to undergop53-dependent cell cycle arrest and/or cell death in response to variousstresses such as DNA damage, hypoxia, and/or exposure to variouschemotherapeutic agents (e.g., microtubule inhibitors) known in the artto induce p53-dependent apoptosis in somatic cells. In some embodimentsthe p53-dependent pathway leads to apoptosis. In some embodiments thep53-dependent pathway leads to cell senescence. One of skill in the artwill be able to determine whether cells have a functional p53 pathway,e.g., by exposing cells to conditions known to induce p53-dependent cellcycle arrest or death and determining whether the cells respond in amanner consistent with existence of a functional p53-dependent pathway.In general, noncancerous somatic cells obtained from a mammalian subjectare expected to possess functional p53 pathways.

In certain embodiments of the invention the somatic cells are sensitiveto DNA demethylation. As used herein, a cell is “sensitive to” DNAdemethylation if it displays decreased ability to survive or proliferateunder conditions of reduced DNA methylation. DNA methylation is requiredfor survival of a variety of different somatic cell types, particularlythose that are proliferating. For example, when the Dnmt1 gene isrendered nonfunctional in proliferating fibroblasts by Cre-mediatedrecombination, the cells exhibit progressive DNA demethylation between3-5 days following introduction of a construct from which Cre isexpressed, and die between 5 and 6 days following introduction of theconstruct (Jackson-Grusby, et al.). DNA demethylation is a propertyshared by proliferating somatic cells of diverse types, consistent withthe fact that Dnmt1, 3a, and 3b are essential genes. In contrast, EScells are able to survive and proliferate in the absence of functionalDNMT1 unless induced to differentiate.

In certain embodiments a population of cells of the present invention ischaracterized in that on average the number of methylated cytosines inthe genomic DNA of the cells is reduced by at least 5% relative to thelevel that would exist under “standard conditions”. In some embodimentsthe population of cells is subjected to conditions such that the numberof methylated cytosines in genomic DNA is reduced on average by between5% and 10%, between 10% and 25%, between 25% and 50%, between 50% and75%, between 75% and 95%, or by between 95% and 100%, relative to thelevel that exists under standard conditions. In certain embodiments ofthe invention the average amount of methylation (i.e., the averagenumber of methylated cytosines) of at least 10, 20, 50, or 100 genesand/or genetic elements such as IAP, L1, LINE, or SINE elements orendogenous retroviral elements is reduced in the population of cellsrelative to an otherwise identical population of cells that has not beensubjected to demethylating conditions. In certain embodiments theaverage expression level of Dnmt mRNA, e.g., Dnmt1 mRNA, in thepopulation of cells is less than 50% of its normal level. In someembodiments the average expression level of DNMT protein, e.g., DNMT1protein, in the population of cells is less than 50% of its normallevel.

A cell is said to be “resistant to DNA demethylation” if it is able tosurvive and to proliferate when DNA methylation is reduced to a levelthat would result in cell cycle arrest or cell death in a proliferatingsomatic cell such as a primary fibroblast. In certain embodiments of theinvention a “proliferating cell” is one that would be expected to dividewithin 96 hours if maintained under appropriate culture conditions. Incertain embodiments the proliferating cell would be expected to dividewithin 72 hours if maintained under appropriate culture conditions. Insome embodiments the cell would be expected to divide within 48 hours,or within 24 hours. In other words, if the cell (and its progeny) is/aremaintained in culture under appropriate conditions the total number ofcells would double within 24, 48, 72, or 96 hours. “Appropriate cultureconditions” refers to standard culture conditions known in the art asbeing suitable for a somatic cell type of interest to survive andproliferate. See, e.g., Masters, J. (ed.) Animal Cell Culture: APractical Approach, 3rd ed., Oxford University Press, 2000; Freshey, I.,et al., Culture of Animal Cells: A Manual of Basic Technique, 5th ed.,Wiley-Liss, 2005.

Reduced DNA methylation can be achieved by (a) inhibiting expression oractivity of an endogenous DNA methyltransferase or otherwise inhibitingDNA methylation by an endogenous DNA methyltransferase, e.g., bycontacting a cell with an agent that inhibits expression or activity ofthe endogenous DNA methyltransferase or otherwise inhibits DNAmethylation; (b) expressing an agent that inhibits expression oractivity of an endogenous DNA methyltransferase or otherwise inhibitsDNA methylation by the DNMT in the cell; (c) inhibiting expression oractivity of an endogenous protein other than a DNA methyltransferase,which protein is needed for any step of a biochemical pathway thatprovides a substrate for the transfer of a methyl group to cytosine by aDNA methyltransferase; (d) expressing in the cell an agent that inhibitsexpression or activity of an endogenous protein other than a DNAmethyltransferase, which protein is needed for any step of a biochemicalpathway that provides a substrate for the transfer of a methyl group tocytosine by a DNA methyltransferase; and/or (e) culturing the cell underconditions in which it is deprived of nutrients needed for synthesis ofa substrate for the transfer of a methyl group to cytosine by a DNAmethyltranferase (but in the presence of sufficient nutrients tootherwise support cell viability). Expressing an agent in the cell asdescribed in (b) or (d) can be achieved by contacting the cell with aagent that induces or derepresses such expression or otherwise causessuch expression (e.g., by a recombinase-mediated mechanism). A cell thathas been treated in any of the afore-mentioned ways (or any other wayknown in the art) to reduce DNA demethylation is said to have beensubjected to “DNA demethylating conditions”. For example, a cell thathas been contacted with an agent that induces expression of an RNAiagent that inhibits DNMT expression or has been contacted with a DNAmethyltransferase inhibitor or a recombinase that inactivates a DNMTgene has been subjected to DNA demethylating conditions.

The DNA methyltransferase can be DNMT, 3a, and/or 3b. In someembodiments expression and/or activity of only DNMT1 is inhibited. Inother embodiments expression and/or activity of DNMT1 and either DNMT3aor 3b is inhibited. In some embodiments expression and/or activity ofDNMT1, 3a, and 3b are inhibited. In some embodiments the endogenousprotein other than a DNMT is an endogenous transporter or enzyme neededfor any step of a biochemical pathway that provides a substrate for thetransfer of a methyl group to cytosine by a DNA methyltransferase in thecell. In some embodiments a combination of conditions is used, e.g., atleast one DNMT is inhibited and cells are cultured in conditions lackingat least some of the nutrients needed for DNA methylation. In anotherembodiment cells are contacted with a small molecule that inhibits DNAmethylation (such as 5′aza-cytidine) and an RNAi agent that inhibitsexpression of DNMT1, 3a, or 3b is expressed in the cells. In certainembodiments a cell that is sensitive to DNA demethylation undergoes cellcycle arrest or death when DNA methylation is reduced and/or when DNAmethyltransferase activity is inhibited, leading to DNA demethylation.

A variety of DNA methylation inhibitors are known in the art and are ofuse in the invention. See, e.g., Lyko, F. and Brown, R., JNCI Journal ofthe National Cancer Institute, 7(20):1498-1506, 2005. Inhibitors of DNAmethylation include nucleoside DNA methyltransferase inhibitors such as5-azacytidine, 5-azadeoxycytidine, and zebularine, non-nucleosideinhibitors such as the polyphenol (−)-epigallocatechin-3-gallate (EGCG)and the small molecule RG108(2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-3-(1H-indol-3-yl)propanoicacid), compounds described in WO2005085196 and phthalamides,succinimides and related compounds as described in WO2007007054. Threeadditional classes of compounds are: (1) 4-Aminobenzoic acidderivatives, such as the antiarrhythmic drug procainamide and the localanesthetic procaine; (2) the psammaplins, which also inhibits histonedeacetylase (Pina, I. C., J Org Chem., 68(10):3866-73, 2003); and (3)oligonucleotides, including siRNAs, shRNAs, and specific antisenseoligonucleotides, such as MG98. DNA methylation inhibitors may act by avariety of different mechanisms. The nucleoside inhibitors aremetabolized by cellular pathways before being incorporated into DNA.After incorporation, they function as suicide substrates for DNMTenzymes. The nonnucleoside inhibitors procaine,epigallocatechin-3-gallate (EGCG), and RG108 have been proposed toinhibit DNA methyltransferases by masking DNMT target sequences (i.e.,procaine) or by blocking the active site of the enzyme (i.e., EGCG andRG108). In some embodiments of the invention combinations of DNAmethylation inhibitors are used. In some embodiments the concentrationsare selected to minimize toxic effects on cells. In some embodimentsagents that incorporate into DNA (or whose metabolic productsincorporate into DNA) are not employed. In certain embodiments of theinvention DNA methylation in a cell is considered “reduced”, and the DNAof the cell is considered at least in part “demethylated”, if the numberof methylated cytosines in the cell's genomic DNA is reduced by at least5% relative to the level that would exist under “standard conditions”,by which are meant conditions within the body of a mammalian subject orappropriate cell culture conditions known and routinely used in the artfor cells of a particular type of interest. In some embodiments thenumber of methylated cytosines in genomic DNA is reduced by between 5%and 10%, between 10% and 25%, between 25% and 50%, between 50% and 75%,between 75% and 95%, or by between 95% and 100%, relative to the levelthat exists under standard conditions, e.g., prior to administration orinduction of expression of an inhibitor of a DNA methyltransferase. Incertain embodiments of the invention DNA methylation in a cell isconsidered “reduced”, and the DNA of the cell is considered at least inpart “demethylated”, if the number of methylated CpG sequences in thecell's genomic DNA is reduced by at least 5% relative to the level thatwould exist under “standard conditions”, by which are meant conditionswithin the body of a mammalian subject or appropriate cell cultureconditions known and routinely used in the art for cells of a particulartype of interest. In some embodiments the number of methylated CpGsequences in genomic DNA is reduced by between 5% and 10%, between 10%and 25%, between 25% and 50%, between 50% and 75%, between 75% and 95%,or by between 95% and 100%, relative to the level that exists understandard conditions, e.g., prior to administration or induction ofexpression of an inhibitor of a DNA methyltransferase. In certainembodiments the cell is subjected to global DNA demethylation. “GlobalDNA demethylation” refers to DNA demethylation that occurs at manylocations in the genome as opposed to at one or a few specific loci. Incertain embodiments of the invention global DNA demethylation reducesthe methylation (i.e., the number of methylated cytosines) of at least10, 20, 50, or 100 genes and/or genetic elements such as IAP, L1, LINE,or SINE elements or endogenous retroviral elements. One of skill in theart will readily be able to determine qualitatively whether the cell'sDNA is demethylated and/or to determine the extent of demethylation. Forexample, one of skill in the art could make use of the fact that certainrestriction enzymes and/or DNA cleaving agents recognize only methylatedDNA. In certain embodiments bisulfite sequencing is employed. In oneembodiment bisulfite treatment followed by PCR amplification of DNArepetitive elements is employed (Yang, A. S., et al., Nucl. Acids Res.,32(3): e38, 2004). In certain embodiments HPLC or nearest neighboranalysis is used to quantify the amount of 5-methylcytosine.

In some embodiments cell cycle arrest or death occurs within 30 days orless following subjecting the cells to demethylating conditions, e.g.,within 15 days or less, within 10 days, within 5 days, etc. In someembodiments cell cycle arrest or death occurs within 5-6 days followingsubjecting the cells to demethylating conditions. In some embodiments,cell cycle arrest or death occurs within 10 times the time required forthe cell to complete 10 cell cycles under non-demethylating conditions,e.g., between 5-10 cell cycle times or between 2-5 cell cycle timesfollowing subjecting the cells to demethylating conditions. In someembodiments cell cycle arrest or death occurs within 30 days or lessfollowing inducing expression of an RNAi agent targeted to a Dnmt genein the cells, e.g., within 15 days or less, within 10 days, within 5days, etc. In some embodiments cell cycle arrest or death occurs within5-6 days following inducing expression of an RNAi agent targeted to aDnmt gene in the cells. In some embodiments, cell cycle arrest or deathoccurs within 10 times the time required for the cell to complete 10cell cycles under conditions in which the Dnmt gene is expressed normal,e.g., between 5-10 cell cycle times or between 2-5 cell cycle timesfollowing inducing the expression of an RNAi agent targeted to a Dnmtgene in the cells. In some embodiments cell cycle arrest or death occursafter 30 days or less during which the average expression level of DnmtmRNA, e.g., Dnmt1 mRNA, is less than 50% of its normal level, e.g.,after 15 days or less, after 10 days or less, or after 5 days or less.In some embodiments cell cycle arrest or death occurs after 30 days orless during which the average expression level of DNMT protein, e.g.,DNMT1 protein, is less than 50% of its normal level, e.g., within 15days or less, after 10 days, after 5 days, etc. In some embodiments cellcycle arrest or death occurs after 30 days or less during which theaverage methyltransferase activity level of DNMT protein, e.g., DNMT1protein, is less than 50% of its normal level, e.g., after 15 days orless, after 10 days or less, or after 5 days or less.

It will be appreciated that the methods of the invention are oftenpracticed using populations of somatic cells. A population of somaticcells is said to be sensitive to DNA demethylation if at least 90% ofthe cells are sensitive to DNA demethylation. In some embodiments atleast 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9, 99.95% or more of thecells are sensitive to DNA demethylation. Thus when the cells aresubjected to demethylating conditions, at least at least 95%, 96%, 97%,98%, 99%, 99.5%, 99.8%, 99.9%, 99.95% or more of the cells undergo cellcycle arrest or die within a selected time period, e.g., within 30 days,within 15 days, within 10 days, etc. The population of cells may be of asingle type and may be substantially free of other cell types.“Substantially free” as used herein refers to at least about 80% pure,preferably 85%, 90%, 95%, 99% or more pure population of the desiredcells in the whole cell population. In some embodiments the cells arecultured in medium that supports growth of only a desired cell type fora period of time, thereby resulting in a population of cellssubstantially free of other cell types.

In certain embodiments of the invention, reprogrammed somatic cells areidentified by a method that comprises selecting for cells that areresistant to DNA demethylation. The invention provides a method ofidentifying a somatic cell that has been at least in part reprogrammedto an ES-like state, the method comprising steps of: (a) providingsomatic cells, at least some of which have been at least in partreprogrammed to an ES-like state; and (b) selecting a cell that isresistant to DNA demethylation, thereby identifying a cell that has anincreased likelihood of having been reprogrammed, e.g., reprogrammed toan ES-like state. In some embodiments at least some of the cellsidentified using the method have been reprogrammed to an ES-like state.In some embodiments at least some of the cells have been at least inpart reprogrammed to an ES-like state, such that they are moresusceptible to reprogramming to a pluripotent state when subjected toone or more additional treatments than cells that are not resistant toDNA demethylation.

The method makes use of the fact that many or most somatic cell typesare sensitive to DNA demethylation, i.e., they cannot survive orproliferate for prolonged periods of time without the ability tomaintain sufficient methylation of their genomic DNA. In contrast, EScells are resistant to DNA demethylation and can survive in the absenceof endogenous DNA methyltransferase. In some embodiments a population ofsomatic cells is subjected to conditions under which at least 70%, atleast 80%, or at least 90% of unreprogrammed somatic cells of that celltype would be expected to cease proliferating or to die within 30 daysafter being subjected to the conditions. In some embodiments at least90% of unreprogrammed somatic cells of that type would be expected tocease proliferating or die within 20 days after being subjected to theconditions. In some embodiments a population of somatic cells issubjected to conditions under which at least 95% of unreprogrammedsomatic cells of that cell type would be expected to cease proliferatingor to die within 15 days after being subjected to the conditions. Inanother embodiment a population of somatic cells is subjected toconditions under which at least 99% of unreprogrammed somatic cells ofthat cell type would be expected to cease proliferating or to die within10 days after being subjected to the conditions. In some embodiments thecells are human cells. In some embodiments the somatic cells areproliferating cells. In some embodiments the somatic cells arefibroblasts. In certain embodiments the somatic cells express anexogenously introduced reprogramming factor. In certain embodiments thecells have been contacted with a reprogramming agent. In someembodiments the cells are subjected to conditions under which DNA isdemethylated. In certain embodiments the somatic cells reversiblyexpress an RNAi agent targeted to an endogenous DNA methyltransferase.In certain embodiments, the method further comprises after the cell isselected, inhibiting (i.e., reducing or eliminating) the expression ofthe RNAi agent in the selected somatic cell, thereby allowing thegenomic DNA of the selected somatic cell to become methylated. Thus DNAmethylation can occur as the cell is maintained in culture and/or as itsprogeny are induced to differentiate.

The invention further provides a method of identifying a somatic cellthat has been at least in part reprogrammed to a pluripotent state, themethod comprising providing somatic cells that are sensitive to DNAdemethylation; contacting the cells with one or more factors capable ofreprogramming somatic cells; treating the cells so as to reducemethylation of genomic DNA; maintaining the cells in culture for a timeperiod; and identifying a cell that is alive after said time period,thereby identifying a cell that has an increased likelihood of havingbeen at least in part reprogrammed to a pluripotent state. In someembodiments at least some of the cells identified using the method havebeen reprogrammed to an ES-like state. In some embodiments at least someof the cells have been at least in part reprogrammed to an ES-likestate, such that they are more susceptible to reprogramming to apluripotent state when subjected to one or more additional treatmentsthan cells that are not resistant to DNA demethylation. In certainembodiments of the invention the cells are then subjected to suchadditional treatment(s). One of skill in the art will be able to test apopulation of somatic cells to determine the conditions and the timeperiod needed such that a desired fraction of the cells in a populationwill not survive when subjected to demethylating conditions.

For example, one may culture the cells after inducing expression of anRNAi agent targeted to the Dnmt1 gene and count the number of viablecells at different time points to determine the length of time (“X”hours or days) needed for at least 80%, at least 90%, at least 95%, orat least 99% of the cells to be killed as a consequence of reduced DNAmethylation. When practicing the inventive methods, cells that have beentreated with an agent capable of reprogramming cells and are viableafter X hours or days are potentially reprogrammed. It will beappreciated that not all viable cells may be reprogrammed. For example,some of the cells may not express the RNAi agent at levels sufficient tokill unreprogrammed cells. The cells may be subjected to one or moreadditional selections or tests to determine whether they arereprogrammed or to select from the potentially reprogrammed cells thosethat are reprogrammed. For example, cells that are viable after time Xmay be subjected to a screen or selection for cells that have twotranscriptionally active X chromosomes (in the case of cells derivedfrom a female), and/or may be screened or selected for cells thatexpress one or more markers characteristic of ES cells, etc.

The invention further provides a method of identifying a differentiatedsomatic cell that has been reprogrammed to a pluripotent state, themethod comprising providing a population of cells, at least some ofwhich have been reprogrammed to a pluripotent state, wherein said cellcomprises a polynucleotide encoding a selectable marker operably linkedto expression control elements that regulate expression of an endogenouspluripotency gene in such a manner that expression of the selectablemarker substantially matches expression of the endogenous pluripotencygene, and identifying a cell that expresses the selectable marker,thereby identifying a somatic cell that has an increased likelihood ofhaving been reprogrammed to a pluripotent state (relative to cells thatdo not express the selectable marker). In some embodiments, theendogenous pluripotency gene is Oct-4 or Nanog. In some embodiments themethod further comprises selecting a cell or colony of cells having amorphology characteristic of an ES cell or ES cell colony. Morphologicalcriteria known in the art can be used to select such cells or colonies.

In a further embodiment of the invention, reprogrammed somatic cells areidentified by selecting for cells that contain two transcriptionallyactive X chromosomes. In one embodiment the invention provides a methodof identifying a somatic cell that has an increased likelihood of havingbeen reprogrammed to an ES-like state, the method comprising providingsomatic cells that contain two X chromosomes, one of which is inactive;subjecting the cells to one or more treatments that reprogram somaticcells; and identifying a cell in which the inactive X chromosome hasbecome active, thereby identifying a cell that has an increasedlikelihood of having been reprogrammed, e.g., reprogrammed to an ES-likestate. In some embodiments at least some of the cells identified usingthe method have been reprogrammed to an ES-like state. In someembodiments at least some of the cells have been at least in partreprogrammed to an ES-like state, such that they are more susceptible toreprogramming to a pluripotent state when subjected to one or moreadditional treatments than cells that do not have two transcriptionallyactive X chromosomes. In certain embodiments of the invention the cellsare then subjected to such additional treatment(s).

In certain embodiments the somatic cells contain two X chromosomes, oneof which is inactive, wherein one of the X chromosomes contains afunctional allele of a selectable marker gene and the other X chromosomedoes not contain a functional allele of said selectable marker gene. Incertain embodiments the selectable marker gene is an endogenous genenormally present on the X chromosome. In certain embodiments the somaticcells contain two X chromosomes, one of which is inactive, wherein bothof the X chromosomes contain a functional allele of a selectable markergene.

In certain embodiments the somatic cells contain two X chromosomes, oneof which is inactive, wherein both of the X chromosomes contain afunctional allele of a selectable marker gene that is useful for bothpositive and negative selection, and the method comprises: (a) selectingcells that do not express the selectable marker gene, thereby obtaininga population of cells in which a first X chromosome is transcriptionallyinactive; (b) subjecting the cells to one or more treatments thatreprogram the cells; (c) functionally inactivating the selectable markergene on said first X chromosome; and (d) selecting cells that expressthe selectable marker gene, thereby selecting cells in which the secondX chromosome is transcriptionally active. In certain embodiments theselectable marker gene is an endogenous gene normally present on the Xchromosome, e.g., the Hprt gene.

The invention further provides a method of identifying a somatic cellhaving an increased likelihood of having been reprogrammed to an ES-likestate, the method comprising steps of: (a) providing somatic cells thathave an active X chromosome that lacks a functional allele of aselectable marker and an inactive X chromosome that contains afunctional allele of said selectable marker; (b) subjecting the cells toone or more treatments capable of reprogramming somatic cells; and (c)selecting cells that express the selectable marker gene, therebyselecting cells in which the inactive X chromosome has becometranscriptionally active. Such cells have an increased likelihood ofhaving been reprogrammed to an ES-like state relative to cells in whichthe inactive X chromosome has not become transcriptionally active.

The invention further provides a method of identifying a somatic cellthat has an increased likelihood of having been reprogrammed to anES-like state, the method comprising: (a) providing somatic cells thatcontain two X chromosomes, one of which is inactive, wherein one of theX chromosomes contains a functional allele of a selectable marker geneand the other X chromosome does not contain a functional allele of theselectable marker gene; (b) selecting cells that do not express theselectable marker gene, thereby selecting cells in which the inactive Xchromosome contains a functional allele of the selectable marker gene;(c) subjecting the cells to one or more treatments capable ofreprogramming somatic cells; and (d) selecting cells that express theselectable marker gene, thereby selecting cells in which the inactive Xchromosome has become transcriptionally active. Such cells have anincreased likelihood of having been reprogrammed to an ES-like staterelative to cells in which the inactive X chromosome has not becometranscriptionally active.

Somatic cells that have a first X chromosome that lacks a functionalallele of a selectable marker can be prepared in a variety of ways. Forexample, homologous recombination could be used to delete all or part ofthe allele. Cells in which the allele was successfully inactivated canbe selected using conventional methods. Alternatively, the cells may notbe genetically engineered but may instead harbor a mutation thatinactivates the gene. The cells may have been exposed to a mutagen orcondition such as UV radiation to increase the proportion of cellshaving such a mutation or the mutation may spontaneously arise underselective pressure. In one embodiment, the selectable marker is one thatis usable for positive and negative selection such as Hprt. In suchembodiments cells in which one X chromosome lacks a functional allele ofthe gene are selected under conditions that select against cells thatexpress the marker. For example in the case of Hprt, cells may beselected by culturing them in medium containing thioguanine. Aftersubjecting the cells to a treatment capable of reprogramming somaticcells, cells that express the marker are selected, e.g., by culturing inHAT medium. Such cells will have reactivated the inactive X chromosomeand thus have an increased likelihood of having been reprogrammed. Atleast some of the cells identified using the method are reprogrammedsomatic cells.

Certain methods of the invention include a step of selecting cells thatexpress a marker that is expressed by multipotent or pluripotent cells.The marker may be specifically expressed in such cells. One couldculture potentially reprogrammed cells in the presence of antibodiesthat have a detectable label attached thereto to and use flow cytometry(e.g., fluorescence activated cell sorting) to separate cells thatexpress the marker (indicative of a reprogrammed state) from cells thatdo not. In other embodiments, an affinity-based separation method isused to separate reprogrammed cells from cells that are notreprogrammed. In one embodiment, reprogrammed somatic cells are selectedby contacting the cells with a solid or semi-solid support that has abinding agent that specifically binds to an ES cell surface markerattached thereto. The support has a surface to which a binding agent canbe bound. The surface could comprise, e.g., plastic (polypropylene,polyvinyl chloride, polyvinylidene chloride, polytetrafluorethylene,polyethylene, polyamides), glass, metal (e.g., silicon), agarose, etc.Useful supports include agarose or agarose-based matrices (e.g., agaroseor sepharose beads), particles that consist at least in part of amagnetic material, particles comprising polymers such as styrene orlatex, tissue culture vessels or plates, tubes (e.g., microfuge tubes),membranes, etc. In some embodiments the support is a population ofparticles such as magnetic beads. Such particles are often under 100microns in longest axial dimension, e.g., between 1 and 10 microns, andoften approximately spherical. Magnetic beads and methods of using themfor cell separation are known in the art and are commercially availablefrom many sources. For example, Dynabeads (Dynal Biotech, Norway) aresuperparamagnetic polymer beads that have a dispersion of magneticmaterial throughout with a thin polymer shell. Binding agents can becovalently or noncovalently attached to the surface using conventionalmethods. The binding agent could be a naturally occurring or artificialpeptide or polypeptide, small molecule, nucleic acid (e.g., an aptamer),that specifically binds to the ES cell surface marker. In one embodimentthe binding agent is an antibody or antibody fragment. In anotherembodiment the binding agent is a ligand for a receptor. In someembodiments cells are incubated in a liquid medium in the presence ofmagnetic beads that have a binding agent attached thereto. A magneticforce is used to retrieve the beads from the medium. For example, thebeads may be attracted to the side of a vessel and the medium removed.Cells are recovered from the beads, or the beads are removed from thecells, using standard methods such as competition with the binding agentor by contacting the beads with an affinity reagent that binds to amolecule present on the surface of the beads but does not bind to thecells.

Alternately or additionally, one could select cells that do not expressmarkers characteristic of somatic cells from which the potentiallyreprogrammed cells were derived and which are not expressed in ES cellsgenerated using conventional methods. For example, one could incubatecells in the presence of a first binding agent (e.g., an antibody) thatbinds to a marker characteristic of a somatic cell and not found on apluripotent cell. If the binding agent is labeled, flow cytometry couldbe used to isolate cells that do not have the antibody attached thereto.In another embodiment, a second binding agent that binds to the firstthe binding agent is used to remove cells that have the first bindingagent bound thereto. In another embodiment the first binding agent iscrosslinked and precipitated to remove cells that express a markercharacteristic of somatic cells. Other methods of separating cells mayutilize differences in average cell size or density that may existbetween pluripotent cells and somatic cells. For example, cells can befiltered through materials having pores that will allow only certaincells to pass through.

The methods of the invention may be combined in any order. In someembodiments cell that express a first ES cell marker are selected, andthe cells are then assessed for an additional pluripotencycharacteristic such as expression of a second ES cell marker, resistanceto DNA methylation, having two transcriptionally active X chromosomes,etc. In some embodiments cell that are resistant to DNA methylationand/or have two transcriptionally active X chromosomes are selected,thereby providing a population of cells enriched for reprogrammed cells.The cells are then subjected to an additional enrichment step comprisingselecting cells that express a first ES cell marker. Optionally thecells are then tested to determine whether they express a second ES cellmarker. Any number of markers may be used to enrich for ES-like cellsand/or their expression assessed.

The invention thus allows the artisan to prepare a purified preparationof pluripotent reprogrammed somatic cells. Somatic cells may bereprogrammed to gain either a complete set of the pluripotencycharacteristics and are thus pluripotent. Alternatively, somatic cellsmay be reprogrammed to gain only a subset of the pluripotencycharacteristics. In another alternative, somatic cells may bereprogrammed to be multipotent.

The instant specification provides a number of methods to identifyand/or select reprogrammed cells, wherein the cells have a geneticmodification usable for such purposes and/or wherein a chemical orgenetic selection based on such genetic modification is employed.However, as described herein, somatic cells that have been reprogrammedto an ES-like state can be identified without use of such chemical orgenetic selection. Thus the invention further provides methods ofderiving reprogrammed somatic cells from somatic cells that are notgenetically modified, and further provides reprogrammed somatic cellsderived using the inventive methods. In some embodiments somatic cellsthat are not genetically modified can be obtained from a variety ofspecies. For example, suitable cells can be obtained from mice, rats,rabbits, farm animals (e.g., sheep, goats, horses, cows and the like),companion animals (e.g., dogs, cats and the like), primates and humansand used to derive ES-like pluripotent or multipotent cells. In someembodiments the methods employ morphological criteria to identifycolonies containing reprogrammed somatic cells from a population ofcells. The colonies are subcloned and/or passaged once or more incertain embodiments, thereby obtaining a population of cells enrichedfor ES-like cells. The enriched population may contain at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or more, e.g., 100% ES-like cells. Theinvention provides cell lines of somatic cells that have been stably andheritably reprogrammed to an ES-like state.

“Genetic selection” encompasses methods in which genetic material (e.g.,DNA) is introduced into cells, wherein introduced genetic materialallows desired cells (e.g., cells having one or more desiredcharacteristics) to be distinguished from other cells. For example, anendogenous pluripotency gene linked to DNA encoding a detectable markersuch as a fluorescent protein, would allow genetic selection. “Chemicalselection” encompasses methods that involve exposing cells to a chemicalagent that exerts negative selective pressure on undesired cells, e.g.,kills them or reduces their rate of proliferation and/or allows onlydesired cells to survive and/or proliferate. For example, an endogenouspluripotency gene linked to DNA encoding a drug resistance marker suchas neo, would allow chemical selection by culturing cells in thepresence of a chemical agent (e.g., G418) that kills cells notexpressing the drug resistance marker. Such selection would also beconsidered a genetic selection since it makes use of introduced geneticmaterial. In some embodiments, a chemical selection method is employed,but the method does not depend on the presence of genetic material notnaturally found in the cell. For example, the chemical selection may bedirected against a naturally occurring cell product, e.g., a cellsurface marker. In some embodiments the chemical selection method doesnot employ an antibiotic.

The invention provides methods of deriving reprogrammed somatic cellsfrom somatic cells without requiring genetic modification of the cellsthat are to be reprogrammed. In some embodiments, the reprogrammedsomatic cells do not contain exogenous genetic material introduced intothe genome of said cells (or ancestors of said cells) by the hand ofman. In some embodiments the reprogrammed somatic cells do not containgenetic material introduced either transiently into the cells orintroduced stably into the genome of said cells (or ancestors of saidcells) by the hand of man. In some embodiments, cells are transientlytransfected with a construct that encodes a protein that contributes toreprogramming, wherein the construct encodes a drug resistance marker orother selectable marker. Selective pressure is maintained for asufficient period of time for the cells to become reprogrammed.Subsequently, after a sufficient period of time for the cells to becomeat least in part reprogrammed and/or to activate endogenous pluripotencygene(s) such as Oct4, a second selection is applied to select cells thathave lost the construct. In some embodiments the reprogrammed somaticcells do contain exogenously introduced genetic material in theirgenome, but such genetic material is introduced for purposes of (i)inducing the reprogramming process and/or (ii) correcting a geneticdefect in such cells or enabling such cells to synthesize a desiredprotein for therapeutic purposes and, in either case, is not used toselect reprogrammed cells. It will be appreciated that geneticmodifications performed in order to induce reprogramming are distinctfrom genetic modifications whose purpose is to allow selection ofreprogrammed cells and does not itself contribute to reprogramming.

In some embodiments, the methods employ morphological criteria toidentify reprogrammed somatic cells from among a population of somaticcells that are not reprogrammed. In some embodiments, the methods employmorphological criteria to identify somatic cells that have beenreprogrammed to an ES-like state from among a population of cells thatare not reprogrammed or are only partly reprogrammed to an ES-likestate. “Morphological criteria” is used in a broad sense to refer to anyvisually detectable aspect of the size, shape, structure, organization,and/or physical form of the cells or colonies. Identification based onmorphological is distinct from identification based on visuallydetectable expression of a particular selectable marker (e.g., afluorescent protein) by the cells. Morphological criteria include, e.g.,the shape of the colonies, the sharpness of colony boundaries (withsharp boundaries characterizing colonies of ES-like cells), the densityof the cells in the colonies (with increased density characterizingcolonies of ES-like cells), and/or the small size and distinct shape ofthe reprogrammed cells relative to non-reprogrammed cells, etc. Theinvention encompasses identifying and, optionally, isolating colonies(or cells from colonies) wherein the colonies display one or more suchcharacteristics depicted in these figures.

The reprogrammed somatic cells may be identified as colonies growing ina first tissue culture dish, and the colonies, or portions thereof,transferred to a second tissue culture dish, thereby isolatingreprogrammed somatic cells. “Tissue culture dish” as used herein refersto any vessel, plate, receptacle, container, etc., in which living cellscan be maintained in vitro. The bottom of the tissue culture dish may beat least in part coated with a substrate, e.g., a protein or mixturethereof such as gelatin, Matrigel, fibronectin or other cell adhesionmolecule, collagen, protein-based or non-protein based hydrogel, etc.,on which the cells are disposed. In some embodiments the dish contains afeeder cells (optionally irradiated), which may at least in part coatthe bottom of the dish.

In some embodiments, the methods employ complement-mediated lysis toeliminate at least some non-reprogrammed somatic cells from a populationof cells that contains at least some reprogrammed somatic cells. In oneembodiment, a population of somatic cells is contacted with acomplement-fixing antibody (e.g., an IgG or IgM antibody) that binds toa cell surface marker that is not detectably expressed by pluripotentcells, e.g., ES cells (or is expressed at much lower, e.g.,insignificant levels by such cells) but is expressed by unreprogrammedsomatic cells (e.g., unreprogrammed fibroblasts). Such lower levels maybe, e.g., less than 20%, less than 10%, less than 5%, or less than 1%the average level of expression found in unreprogrammed cells in variousembodiments of the invention, or such level as will not be sufficient tosupport complement-mediated lysis of a majority of the cells. The cellsare further contacted with complement components (“complement”)sufficient to mediate lysis of the cells to which the antibody is bound.In one embodiment the cells are contacted with serum, e.g., mouse orhuman serum containing complement. In one embodiment the cells arecontacted with recombinant complement components (e.g., complementcomponents sufficient to mediate the classical pathway such as C1, C2,C3, C4, C5, and C6-C9). Cells that survive in the presence of complementand the antibody are identified as having an increased likelihood ofbeing reprogrammed. The method is of use to enrich or select forreprogrammed cells. In one embodiment, the cell surface marker is an MHCClass I antigen (“MHC”). For example, as shown in Example 9, mouse cellsthat have been reprogrammed to an ES-like state (iPS cells) turn offMHC. Cells picked randomly after transduction with factors that resultin reprogramming and sorted for Oct4 activation are MHC negative.Furthermore, MHC negative cells in a cell population transduced with thefactors are more likely to be reprogrammed. Infected cells sorted forMHC negative: many more colonies than in high MHC population.Complement-mediated depletion (killing of un-reprogrammed cells) leadsto enrichment of SSEA1 positive cells. Complement-mediated selectionleads to much higher number of colonies exhibiting morphologicalfeatures indicative of reprogramming.

In some embodiments of the invention two or more methods, neither ofwhich employs genetic or chemical selection, are employed. For example,the invention provides a method of deriving reprogrammed cellscomprising steps of: (i) providing a population of non-geneticallymodified cells, at least some of which are partly or fully reprogrammedto an ES-like state; (ii) enriching for partly or fully reprogrammedcells using complement-mediated lysis to eliminate at least someunreprogrammed cells; and (iii) identifying reprogrammed cells orcolonies comprising such cells using morphological criteria.

Any of the methods of the invention that relate to generating,selecting, or isolating a reprogrammed somatic cell may include a stepof obtaining a somatic cell or obtaining a population of somatic cellsfrom a donor in need of cell therapy. Reprogrammed somatic cells aregenerated, selected, or identified from among the obtained cells orcells descended from the obtained cells. Optionally the cell(s) areexpanded in culture prior to generating, selecting, or identifyingreprogrammed somatic cell(s) genetically matched to the donor.

Methods for Screening for an Agent that Reprograms Somatic Cells

The present invention also provides methods for identifying an agentthat reprograms somatic cells to a less-differentiated state, as well asthe agents thus identified. In one embodiment, the methods comprisecontacting the engineered or selected somatic cells of the inventionwith a candidate agent, selecting for cells that express the appropriateselectable marker. The presence of cells that express the appropriateselectable marker indicates that the agent reprograms somatic cells.Such an agent is referred to as a “reprogramming agent” or “an agentthat reprograms cells” for purpose of this application. In someembodiments of the invention the reprogramming agent is not Sox2, Oct4,c-myc, Klf4 or Nanog.

In a further embodiment, the methods comprise contacting the engineeredsomatic cells of the invention with a candidate agent, selecting forcells that express the appropriate selectable marker, and assessing thecells so selected for pluripotency characteristics. The presence of acomplete set of pluripotency characteristics indicates that the agentreprograms somatic cells to become pluripotent.

In a further embodiment the invention provides a method of identifyingan agent that reprograms somatic cells to a less differentiated state,the method comprising steps of: (a) contacting somatic cells with acandidate reprogramming agent, wherein the somatic cells are sensitiveto reduced DNA methylation; and (b) determining whether more of thecells are resistant to reduced DNA methylation than would be expected ifthe agent does not reprogram somatic cells, wherein the candidatereprogramming agent is identified as a reprogramming agent if more ofthe cells are resistant to reduced DNA methylation than would beexpected if the candidate reprogramming agent does not reprogram somaticcells. In certain embodiments the method comprises maintaining the cellsin culture under conditions of reduced DNA methylation and determiningwhether more of the cells survive than would be expected if the agentdoes not reprogram somatic cells. In certain embodiments of theinvention the cells are proliferating cells, i.e., they are notpost-mitotic.

In a further embodiment the invention provides a method of identifyingan agent that reprograms somatic cells to a less differentiated state,the method comprising steps of: (a) contacting somatic cells with acandidate reprogramming agent, wherein the somatic cells are sensitiveto reduced DNA methylation; and (b) determining the amount of cells thatare resistant to reduced DNA methylation, wherein an increased amount ofcells that are resistant to reduced DNA methylation, as compared to acontrol, is indicative of the candidate agent being a reprogrammingagent. The control may be a parallel sample that has not been treatedwith the candidate agent, or which has been treated with a candidatehaving a known effect (e.g., a positive effect, a negative effect, or noeffect). Alternatively, the control may be a predetermined value for aparticular assay.

The cells may be treated so as to reduce methylation of genomic DNA,e.g., by inhibiting expression of a DNA methyltransferase and/or bycontacting the cells with an agent that inhibits DNA methyltransferaseactivity or otherwise inhibits any step in the pathway leading to DNAmethylation. Suitable methods and agents are described above. In oneembodiment, DNA methylation is reduced by reversibly inducing expressionof an interfering RNA in the cells, wherein the interfering RNA inhibitsexpression of a DNA methyltransferase such as DNMT1. In someembodiments, expression of Dnmt (e.g., Dnmt1) mRNA is reduced in thecells on average by at least 50%, at least 90%, or more. In someembodiments, expression of a DNMT protein, e.g., a DNMT1 protein, isreduced in the cells on average by at least 50%, at least 90%, or more.Engineered somatic cells useful for practicing the methods are describedabove.

The cells may be maintained in culture for a period of time after beingcontacted with the candidate reprogramming agent but before subjectingthe cells to conditions under which DNA demethylation occurs. Forexample, the cells may be maintained in the presence of the candidatereprogramming agent for between 1 and 12 hours, between 12 and 24 hours,between 24 and 48 hours, between 48 and 72 hours, etc., prior tosubjecting the cells to DNA demethylating conditions. Alternately thecells can be contacted with the agent after the DNA demethylatingconditions have been imposed, e.g., up to 1, 2, 5, or 10 days after DNAdemethylating conditions have been imposed. The candidate reprogrammingagent may, but need not be, present while the cells are subjected toconditions under which DNA demethylation occurs. The cells may bemaintained in culture under conditions of reduced DNA methylation, e.g.,under conditions in which expression of one or more endogenous DNMTproteins is reduced. If cells are able to survive and/or proliferateunder such conditions in greater numbers than would be expected if thecells are not reprogrammed, then the agent is identified as one thatreprograms somatic cells. The cells may be maintained in culture for,e.g., at least 5 days, up to 10 days, up to 15 days, up to 30 days,etc., under conditions of reduced DNA methylation. In some embodimentsthe agent is identified as an agent that reprograms cells if there areat least 2, 5, or 10 times as many viable cells after said time periodif the cells have been contacted with the candidate agent than if thecells have not been contacted with the agent.

The presence of living cells can be assessed using any method known inthe art for assessing cell viability. For example, the ability of thecells to exclude a dye, ability of cells to carry out an enzymaticreaction, MTT assay, measuring incorporation of a labeled substrate, orvisual observation under a microscope are examples of methods that canbe used to determine whether there are living cells and to quantifythem. In some embodiments viable cells produce a fluorescent orluminescent signal. In some embodiments, the assay comprises determiningwhether the cells are undergoing apoptosis. For example, expression ofgenes that induce or participate in apoptosis such as caspases can beassessed, or an assay that examines DNA fragmentation can be used.

In some embodiments the method further comprises determining whether thecells have an intact p53 pathway, such that the cells could underp53-dependent apoptosis and/or cell cycle arrest. In some embodimentscells that are resistant to DNA demethylation but are still able toundergo p53-dependent apoptosis are selected. Thus in certainembodiments the candidate agent is not one that inhibits p53 or a generequired for cells to undergo p53-dependent apoptosis.

The invention further provides a method of identifying an agent thatreprograms somatic cells to a less differentiated state, the methodcomprising steps of: (a) providing somatic cells containing two Xchromosomes, one of which is inactive; (b) contacting the somatic cellswith a candidate reprogramming agent; (c) maintaining the cells inculture; (d) determining whether more of the cells reactivate theirinactive X chromosome while in culture than would be expected if thecandidate agent does not reprogram somatic cells, wherein the candidateagent is identified as a reprogramming agent if more of the cellsreactivate their inactive X chromosome than would be expected if thecandidate reprogramming agent does not reprogram somatic cells. In oneembodiment the method comprises steps of: (a) providing somatic cellscontaining two X chromosomes, one of which is inactive, wherein one ofthe X chromosomes contains a functional allele of a selectable markergene and the other X chromosome does not contain a functional allele ofsaid selectable marker gene; (b) selecting cells that do not express theselectable marker, thereby selecting cells in which the X chromosomethat contains the selectable marker gene is inactive; (c) contacting thesomatic cells selected in step (b) with a candidate reprogramming agent;(d) determining whether more of the cells express the selectable markerthan would be expected if the X chromosome that contains the functionalallele of the selectable marker gene remains inactive, therebydetermining whether more of the cells reactivated their inactive Xchromosome than would be expected if the candidate reprogramming agentdoes not reprogram somatic cells; and (e) identifying the candidateagent as a reprogramming agent if more of the cells reactivate theirinactive X chromosome than would be expected if the candidatereprogramming agent does not reprogram somatic cells. In one embodimentthe afore-mentioned method comprises steps of: selecting cells thatexpress a functional form of the selectable marker after contacting thecells with the candidate reprogramming agent, thereby selecting forcells that have reactivated their inactive X chromosome. In certainembodiments of the invention the selectable marker is suitable forpositive selection and negative selection. In certain embodiments themethod comprises maintaining the cells under conditions in which cellsthat express a functional form of the selectable marker substantially donot survive; and after treating the cells with a candidate reprogrammingagent maintaining cells under conditions in which cells that do notexpress a functional form of the selectable marker substantially do notsurvive. In certain embodiments the gene is an endogenous gene presenton the X chromosome, e.g., the gene encodes hypoxanthine-guaninephosphoribosyltransferase (HPRT). In certain embodiments the Xchromosome that lacks a functional allele of said gene contains anengineered genetic modification that inactivates the gene. In certainembodiments the method comprises steps of:

(a) providing somatic cells containing two X chromosomes, one of whichis inactive, wherein one of the X chromosomes contains a functionalallele of a first selectable marker gene whose expression can beselected against and a functional form of a second selectable markergene whose expression can be selected for, and wherein the other Xchromosome lacks a functional allele of each of said genes; (b)selecting cells that do not express a functional form of the firstselectable marker, thereby selecting cells in which the X chromosomethat contains the functional allele is inactive; (c) contacting thesomatic cells with a candidate reprogramming agent; (d) selecting cellsthat express a functional form of the second selectable marker, therebyselecting for cells that have reactivated their inactive X chromosome;(e) determining whether more of the cells reactivated the inactive Xchromosome than would be expected if the candidate reprogramming agentdoes not reprogram somatic cells; and (f) identifying the candidateagent as a reprogramming agent if more of the cells reactivated theirinactive X chromosome than would be expected if the candidatereprogramming agent does not reprogram somatic cells.

Candidate agents used in the invention encompass numerous chemicalclasses, though typically they are organic molecules, including smallorganic compounds (e.g., compounds having a molecular weight equal to orless than 1500 daltons and multiple carbon-carbon bonds). Candidateagents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, nucleic acidsand derivatives, structural analogs or combinations thereof.

Candidate agents may be naturally arising, recombinant or designed inthe laboratory. The candidate agents may be isolated frommicroorganisms, animals, or plants, or may be produced recombinantly, orsynthesized by chemical methods known in the art. In some embodiments,candidate agents are isolated from libraries of synthetic or naturalcompounds using the methods of the present invention. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, includingacylation, alkylation, esterification, amidification, to producestructural analogs.

There are numerous commercially available compound libraries, including,for example, the Chembridge DIVERSet. Libraries are also available fromacademic investigators, such as the Diversity set from the NCIdevelopmental therapeutics program.

The screening methods mentioned above are based on assays performed oncells. These cell-based assays may be performed in a high throughputscreening (HTS) format, which has been described in the art. Forexample, Stockwell et al. described a high-throughput screening of smallmolecules in miniaturized mammalian cell-based assays involvingpost-translational modifications (Stockwell et al., 1999). Likewise,Qian et al. described a leukemia cell-based assay for high-throughtputscreening for anti-cancer agents (Qian et al., 2001). Both referencesare incorporated herein in their entirety.

A reprogramming agent may belong to any one of many differentcategories. For example, a reprogramming agent may be a chromatinremodeling agent. A chromatin remodeling agent may be a protein involvedin chromatin remodeling or an agent known to alter chromatin toward amore open structure, such as a DNA methylation inhibitor or a histonedeacelyation inhibitor. Exemplary compounds include 5-aza-cytidine, TSAand valproic acid. For another example, such an agent may be apluripotency protein, including, for example, Nanog, Oct-4 and Stella.Such an agent may also be a gene essential for pluripotency in at leastsome contexts, including, for example, Sox2, FoxD3, and LIF, and Stat3.See Smith et al. 1988, William et al., 1988, Ihle, 1996, Avilion et al.,2003, and Hanna et al., 2002). It will be appreciated that the candidatereprogramming agent is typically one that is not present in standardculture medium, or if present is present in lower amounts.

It will also be appreciated that a useful reprogramming agent or otherform of reprogramming treatment need not be capable of reprogramming alltypes of somatic cells and need not be capable of reprogramming allsomatic cells of a given cell type. If the treatment results in apopulation enriched for reprogrammed cells relative to the untreatedpopulation (i.e., has a higher proportion of reprogrammed cells than thestarting population), it is of use in the present invention. Forexample, and without limitation, a reprogramming treatment thatreprograms between 0.000001% and 100% of the treated cells is of use.Also, methods that provide a population of somatic cells that isenriched for reprogrammed cells are of use even if a substantialfraction of the cells are not reprogrammed. Cells in such a populationhave an increased likelihood of being reprogrammed cells relative to anotherwise equivalent population of cells that has not been subjected tothe method. Without limitation, and by way of example, a screen orselection that results in a population of cells in which at least 5% ofthe cells are reprogrammed is of use. Without limitation, a method thatresults in a population that is enriched for reprogrammed cells by afactor of 2, 5, 10, 50, 100 or more (i.e., the fraction of reprogrammedcells in the population is 2, 5, 10, 50, or 100 times more than presentin a starting population) is of use. Multiple selection and/or screeningprocedures can be employed to provide populations of cells that areincreasingly enriched for reprogrammed cells.

In one embodiment of the invention, induced pluripotent cells for use inscreening for candidate reprogramming agents are prepared by a methodcomprising providing one or more somatic cells that each contain atleast one exogenously introduced factor that contributes toreprogramming of said cell to a pluripotent state, wherein each of saidexogenously introduced factors is introduced using an inducible vectorwhich is not subject to methylation-induced silencing and the expressionof which is controlled by regulatory elements induced by distinctinducers (i.e., each exogenously introduced factor is separatelyinducible); (b) maintaining said one or more cells under conditionsappropriate for proliferation of said cells and for activity of said atleast one exogenously introduced factor for a period of time sufficientto reprogram said cell or to activate at least one endogenouspluripotency gene; (c) functionally inactivating said at least oneexogenously introduced factor; (d) selecting one or more cells whichdisplay a marker of pluripotency; (e) generating a chimeric embryoutilizing said one or more cells which display a marker of pluripotency;(f) obtaining one or more somatic cells, e.g., differentiated somaticcells, from said chimeric embryo; (g) maintaining said one or moresomatic cells under conditions appropriate for proliferation of saidcells and for activity of said at least one exogenously introducedfactor for a period of time sufficient to activate at least oneendogenous pluripotency gene; and (h) differentiating between cellswhich display one or more markers of pluripotency and cells which donot. In a preferred embodiment the exogenously introduced factors aresufficient for reprogramming in combination but insufficient if lessthan the combination is expressed. Subcombinations of the exogenouslyintroduced factors can be inducibly expressed, and candidatereprogramming agents, e.g., libraries of agents, can be screened fortheir ability to substitute for the missing factor(s).

In some embodiments of the invention the reprogramming agent is selectedfrom genes encoding Oct-4, Sox-2, c-Myc, and Klf4 and/or the proteinsthemselves. In some embodiments at least 2, 3, or all of said agents areintroduced into somatic cells. One aspect of the invention comprisesmethod of identifying alternate reprogramming agents. For example, 3 ofsaid agents can be introduced into cells, thereby rendering such cellssusceptible to reprogramming. The cells are then used in an inventivescreening method to identify a fourth agent or combination of agentsthat reprograms the cells to an ES-like state. In one embodiment, themethod is used to identify an agent that substitutes for c-myc. In oneembodiment, the method is used to identify an agent that substitutes forKlf4. In one embodiment, the method is used to identify an agent thatsubstitutes for Sox2. In one embodiment, the method is used to identifyan agent that substitutes for Oct-4. In some embodiments the methods arepracticed using human cells and human analogs of the relevant factorsare expressed. In some embodiments the cells are the engineered cells ofthe present invention that contain an endogenous pluripotency genelinked to a selectable marker.

Methods for Gene Identification

The present invention provides methods for identifying a gene thatactivates the expression of an endogenous pluripotency gene in somaticcells. The methods comprise: transfecting the somatic cells of thepresent invention with a cDNA library prepared from ES cells or oocytes,selecting for cells that express the first selectable marker, andassessing the expression of the first endogenous pluripotency gene inthe transfected cells that express the first selectable marker. Theexpression of the first endogenous pluripotency gene indicates that thecDNA encodes a gene that activates the expression of an endogenouspluripotency gene in somatic cells.

The methods are applicable for identifying a gene that activates theexpression of at least two endogenous pluripotency genes in somaticcells. The somatic cells used in the methods further comprise a secondendogenous pluripotency gene linked to a second selectable marker. Themethods are modified to select for transfected cells that express bothselectable markers, among which the expression of the first and thesecond endogenous pluripotency genes are assessed. The expression ofboth the first and the second endogenous pluripotency genes indicatesthat the cDNA encodes a gene that activates the expression of at leasttwo pluripotency genes in somatic cells.

The methods are further applicable for identifying a gene that activatesthe expression of at least three endogenous pluripotency genes insomatic cells. The somatic cells used in the methods further comprise athird endogenous pluripotency gene linked to a third selectable marker.The methods are modified to select for transfected cells that expressall three selectable markers, among which the expression of all threeendogenous pluripotency genes are assessed. The expression of all threeendogenous pluripotency genes indicates that the cDNA encodes a genethat activates the expression of at least three pluripotency genes insomatic cells.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of mouse genetics, developmentalbiology, cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are described in the literature.See, for example, Current Protocols in Cell Biology, ed. by Bonifacino,Dasso, Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons,Inc., New York, 1999; Manipulating the Mouse Embryos, A LaboratoryManual, 3rd Ed., by Hogan et al., Cold Spring Contain Laboratory Press,Cold Spring Contain, New York, 2003; Gene Targeting: A PracticalApproach, IRL Press at Oxford University Press, Oxford, 1993; and GeneTargeting Protocols, Human Press, Totowa, N.J., 2000.

Reprogrammed Somatic Cells and their Uses

The invention thus provides a number of significant advances thatfacilitate therapeutic uses of reprogrammed somatic cells including thefollowing: (i) the ability to reprogram somatic cells lacking geneticmodification to an ES-like state and select such reprogrammed cells froma population of cells that are not reprogrammed or are only partlyreprogrammed to an ES-like state; and (ii) the recognition that stablereprogramming can be achieved by transient presence of reprogrammingagents rather than requiring stable and ongoing expression or exposureto such agents. The first advance allows, among other things, theefficient derivation of ES-like cells from donor-specific somatic cellswithout requiring genetic modification for purposes of selection. Thesecond advance allows, among other things, reprogramming using methodssuch as transient transfection (e.g., of nucleic acid constructsencoding a protein that contributes to reprogramming), proteintransduction, and other methods of introducing agents into cells thatneither require modification of the genome or the introduction of stablyheritable genetic elements into the somatic cells. In summary, theseadvances open the possibility of obtaining donor-specific ES-like cellsby reprogramming somatic cells without the use of genetic modification.

The present invention also provides reprogrammed somatic cells (RSCs),including reprogrammed pluripotent somatic cells (RPSCs), produced bythe methods of the invention. These methods, useful for the generationof cells of a desired cell type, have wide range of applications. Forone example, these methods have applications in livestock management,involving the precise genetic manipulation of animals for economic orhealth purposes. For another example, these methods have medicalapplication in treating or preventing a condition.

Accordingly, the invention provides methods for the treatment orprevention of a condition in a mammal. In one embodiment, the methodsstart with obtaining somatic cells from the individual, reprogrammingthe somatic cells so obtained by methods of the present invention toobtain RPSCs. The RPSCs are then cultured under conditions suitable fordevelopment of the RPSCs into cells of a desired cell type. Thedeveloped cells of the desired cell type are harvested and introducedinto the individual to treat the condition. In an alternativeembodiment, the methods start with obtaining somatic cells from theindividual, reprogramming the somatic cells so obtained by methods ofthe present invention. The RPSCs are then cultured under conditionssuitable for development of the RPSCs into a desired organ, which isharvested and introduced into the individual to treat the condition. Thecondition may be any condition in which cell or organ function isabnormal and/or reduced below normal levels. Thus the inventionencompasses obtaining somatic cells from a donor in need of celltherapy, subjecting the cells to a reprogramming agent such ascontacting the cells with a reprogramming agent, selecting reprogrammedsomatic cells according to a method of the invention. A donor in need ofcell therapy may suffer from any condition, wherein the condition or oneor more symptoms of the condition can be alleviated by administeringcells to the donor and/or in which the progression of the condition canbe slowed by administering cells to the donor.

The RPSCs in certain embodiments of the present invention are ES-likecells, and thus may be induced to differentiate to obtain the desiredcell types according to known methods to differentiate ES cells. Forexample, the RPSCs may be induced to differentiate into hematopoieticstem cells, muscle cells, cardiac muscle cells, liver cells, pancreaticcells, cartilage cells, epithelial cells, urinary tract cells, nervoussystem cells (e.g., neurons) etc., by culturing such cells indifferentiation medium and under conditions which provide for celldifferentiation. Medium and methods which result in the differentiationof embryonic stem cells are known in the art as are suitable culturingconditions.

For example, Palacios et al., Proc. Natl. Acad. Sci., USA, 92: 7530-37(1995) teaches the production of hematopoietic stem cells from anembryonic cell line by subjecting stem cells to an induction procedurecomprising initially culturing aggregates of such cells in a suspensionculture medium lacking retinoic acid followed by culturing in the samemedium containing retinoic acid, followed by transferal of cellaggregates to a substrate which provides for cell attachment.

Moreover, Pedersen, J. Reprod. Fertil. Dev., 6: 543-52 (1994) is areview article which references numerous articles disclosing methods forin vitro differentiation of embryonic stem cells to produce variousdifferentiated cell types including hematopoietic cells, muscle, cardiacmuscle, nerve cells, among others.

Further, Bain et al., Dev. Biol., 168:342-357 (1995) teaches in vitrodifferentiation of embryonic stem cells to produce neural cells whichpossess neuronal properties. These references are exemplary of reportedmethods for obtaining differentiated cells from embryonic or stem-likecells. These references and in particular the disclosures thereinrelating to methods for differentiating embryonic stem cells areincorporated by reference in their entirety herein.

Thus, using known methods and culture medium, one skilled in the art mayculture the subject embryonic or stem-like cells to obtain desireddifferentiated cell types, e.g., neural cells, muscle cells,hematopoietic cells, etc. In addition, the use of inducible Bcl-2 orBcl-xl might be useful for enhancing in vitro development of specificcell lineages. In vivo, BcI-2 prevents many, but not all, forms ofapoptotic cell death that occur during lymphoid and neural development.A thorough discussion of how Bcl-2 expression might be used to inhibitapoptosis of relevant cell lineages following transfection of donorcells is disclosed in U.S. Pat. No. 5,646,008, which is hereinincorporated by reference.

The subject RPSCs may be used to obtain any desired differentiated celltype. Therapeutic usages of such differentiated human cells areunparalleled. For example, human hematopoietic stem cells may be used inmedical treatments requiring bone marrow transplantation. Suchprocedures are used to treat many diseases, e.g., late stage cancerssuch as ovarian cancer and leukemia, as well as diseases that compromisethe immune system, such as AIDS. Hematopoietic stem cells can beobtained, e.g., by fusing adult somatic cells of a cancer or AIDSpatient, e.g., epithelial cells or lymphocytes with an enucleatedoocyte, e.g., bovine oocyte, obtaining embryonic or stem-like cells asdescribed above, and culturing such cells under conditions which favordifferentiation, until hematopoietic stem cells are obtained. Suchhematopoietic cells may be used in the treatment of diseases includingcancer and AIDS.

The methods of the present invention can also be used to treat, prevent,or stabilize a neurological disease such as Alzheimer's disease,Parkinson's disease, Huntington's disease, or ALS, lysosomal storagediseases, multiple sclerosis, or a spinal cord injury. For example,somatic cells may be obtained from the individual in need of treatment,and reprogrammed to gain pluripotency, and cultured to deriveneurectoderm cells that may be used to replace or assist the normalfunction of diseased or damaged tissue.

For the treatment or prevention of endocrine conditions, RPSCs thatproduce a hormone, such as a growth factor, thyroid hormone,thyroid-stimulating hormone, parathyroid hormone, steroid, serotonin,epinephrine, or norepinephrine may be administered to a mammal.Additionally, reprogrammed epithelial cells may be administered torepair damage to the lining of a body cavity or organ, such as a lung,gut, exocrine gland, or urogenital tract. It is also contemplated thatRPSCs may be administered to a mammal to treat damage or deficiency ofcells in an organ such as the bladder, brain, esophagus, fallopian tube,heart, intestines, gallbladder, kidney, liver, lung, ovaries, pancreas,prostate, spinal cord, spleen, stomach, testes, thymus, thyroid,trachea, ureter, urethra, or uterus.

The present invention has the potential to provide an essentiallylimitless supply of isogenic or syngeneic human cells suitable fortransplantation. Such a supply would obviate the significant problemassociated with current transplantation methods, i.e., rejection of thetransplanted tissue which may occur because of host versus graft orgraft versus host rejection. Conventionally, rejection is prevented orreduced by the administration of anti-rejection drugs such ascyclosporin. However, such drugs have significant adverse side-effects,e.g., immunosuppression, carcinogenic properties, as well as being veryexpensive. The present invention may eliminate, or at least greatlyreduce, the need for anti-rejection drugs, such as cyclosporine, imulan,FK-506, glucocorticoids, and rapamycin, and derivatives thereof.

RPSCs may also be combined with a matrix to form a tissue or organ invitro or in vivo that may be used to repair or replace a tissue or organin a recipient mammal. For example, RPSCs may be cultured in vitro inthe presence of a matrix to produce a tissue or organ of the urogenitalsystem, such as the bladder, clitoris, corpus cavernosum, kidney,testis, ureter, uretal valve, or urethra, which may then be transplantedinto a mammal (Atala, Curr. Opin. Urol. 9(6):517-526, 1999). In anothertransplant application, synthetic blood vessels are formed in vitro byculturing reprogrammed cells in the presence of an appropriate matrix,and then the vessels are transplanted into a mammal for the treatment orprevention of a cardiovascular or circulatory condition. For thegeneration of donor cartilage or bone tissue, RPSCs such as chondrocytesor osteocytes are cultured in vitro in the presence of a matrix underconditions that allow the formation of cartilage or bone, and then thematrix containing the donor tissue is administered to a mammal.Alternatively, a mixture of the cells and a matrix may be administeredto a mammal for the formation of the desired tissue in vivo. Preferably,the cells are attached to the surface of the matrix or encapsulated bythe matrix. Examples of matrices that may be used for the formation ofdonor tissues or organs include collagen matrices, carbon fibers,polyvinyl alcohol sponges, acrylateamide sponges, fibrin-thrombin gels,hyaluronic acid-based polymers, and synthetic polymer matricescontaining polyanhydride, polyorthoester, polyglycolic acid, or acombination thereof (see, for example, U.S. Pat. Nos. 4,846,835;4,642,120; 5,786,217; and 5,041,138).

The RPSCs produced according to the invention may be used to producegenetically engineered or transgenic differentiated cells. Essentially,this will be effected by introducing a desired gene or genes, orremoving all or part of an endogenous gene or genes of RPSCs producedaccording to the invention, and allowing such cells to differentiateinto the desired cell type. A preferred method for achieving suchmodification is by homologous recombination because such technique canbe used to insert, delete or modify a gene or genes at a specific siteor sites in the stem-like cell genome.

This methodology can be used to replace defective genes, e.g., defectiveimmune system genes, cystic fibrosis genes, or to introduce genes whichresult in the expression of therapeutically beneficial proteins such asgrowth factors, lymphokines, cytokines, enzymes, etc. For example, thegene encoding brain derived growth factor maybe introduced into humanembryonic or stem-like cells, the cells differentiated into neural cellsand the cells transplanted into a Parkinson's patient to retard the lossof neural cells during such disease. Examples of mutations that may berescued using these methods include mutations in the cystic fibrosisgene; mutations associated with Dunningan's disease such as the R482W,R482Q, and R584H mutations in the lamin A gene; and mutations associatedwith the autosomal-dominant form of Emery Deyfuss muscular dystrophysuch as the R249Q, R453W, and Q6STOP mutations in the lamin A gene. Inthe Q6STOP mutation, the codon for Gln6 is mutated to a stop codon.

Previously, cell types transfected with BDNF varied from primary cellsto immortalized cell lines, either neural or non-neural (myoblast andfibroblast) derived cells. For example, astrocytes have been transfectedwith BDNF gene using retroviral vectors, and the cells grafted into arat model of Parkinson's disease (Yoshimoto et al., Brain Research,691:25-36, (1995)). This ex vivo therapy reduced Parkinson's-likesymptoms in the rats up to 45% 32 days after transfer. Also, thetyrosine hydroxylase gene has been placed into astrocytes with similarresults (Lundberg et al., Develop. Neurol., 139:39-53 (1996) andreferences cited therein).

However, such ex vivo systems have problems. In particular, retroviralvectors currently used are down-regulated in vivo and the transgene isonly transiently expressed (review by Mulligan, Science, 260: 926-932(1993)). Also, such studies used primary cells, astrocytes, which havefinite life span and replicate slowly. Such properties adversely affectthe rate of transfection and impede selection of stably transfectedcells. Moreover, it is almost impossible to propagate a large populationof gene targeted primary cells to be used in homologous recombinationtechniques.

By contrast, the difficulties associated with retroviral systems shouldbe eliminated by the use of RPSCs of the present invention, which areES-like cells. Using known methods to introduced desired genes/mutationsinto ES cells, RPSCs may be genetically engineered, and the resultingengineered cells differentiated into desired cell types, e.g.,hematopoietic cells, neural cells, pancreatic cells, cartilage cells,etc. Genes which may be introduced into the RPSCs include, for example,epidermal growth factor, basic fibroblast growth factor, glial derivedneurotrophic growth factor, insulin-like growth factor (I and II),neurotrophin3, neurotrophin-4/5, ciliary neurotrophic factor, AFT-1,cytokine genes (interleukins, interferons, colony stimulating factors,tumor necrosis factors (alpha and beta), etc.), genes encodingtherapeutic enzymes, collagen, human serum albumin, etc.

In addition, it is also possible to use one of the negative selectionsystems now known in the art for eliminating therapeutic cells from apatient if necessary. For example, donor cells transfected with thethymidine kinase (TK) gene will lead to the production of embryonic(e.g., ES-like) cells containing the TK gene. Differentiation of thesecells will lead to the isolation of therapeutic cells of interest whichalso express the TK gene. Such cells may be selectively eliminated atany time from a patient upon gancyclovir administration. Such a negativeselection system is described in U.S. Pat. No. 5,698,446, and is hereinincorporated by reference. In other embodiments the cells are engineeredto contain a gene that encodes a toxic product whose expression is undercontrol of an inducible promoter. Administration of the inducer causesproduction of the toxic product, leading to death of the cells. Thus anyof the somatic cells of the invention may comprise a suicide gene,optionally contained in an expression cassette, which may be integratedinto the genome. The suicide gene is one whose expression would belethal to cells. Examples include genes encoding diphtheria toxin,cholera toxin, ricin, etc. The suicide gene may be under control ofexpression control elements that do not direct expression under normalcircumstances in the absence of a specific inducing agent or stimulus.However, expression can be induced under appropriate conditions, e.g.,(i) by administering an appropriate inducing agent to a cell or organismor (ii) if a particular gene (e.g., an oncogene, a gene involved in thecell division cycle, or a gene indicative of dedifferentiation or lossof differentiation) is expressed in the cells, or (iii) if expression ofa gene such as a cell cycle control gene or a gene indicative ofdifferentiation is lost. See, e.g., U.S. Pat. No. 6,761,884. In someembodiments the gene is only expressed following a recombination eventmediated by a site-specific recombinase. Such an event may bring thecoding sequence into operable association with expression controlelements such as a promoter. The recombinase may be a differentrecombinase to that used to induce expression of the RNAi agent targetedto a DNA methyltransferase. Expression of the suicide gene may beinduced if it is desired to eliminate cells (or their progeny) from thebody of a subject after the cells (or their ancestors) have beenadministered to a subject. For example, if a reprogrammed somatic cellgives rise to a tumor, the tumor can be eliminated by inducingexpression of the suicide gene. In some embodiments tumor formation isinhibited because the cells are automatically eliminated upondedifferentiation or loss of proper cell cycle control.

Examples of diseases, disorders, or conditions that may be treated orprevented include neurological, endocrine, structural, skeletal,vascular, urinary, digestive, integumentary, blood, immune, auto-immune,inflammatory, endocrine, kidney, bladder, cardiovascular, cancer,circulatory, digestive, hematopoeitic, and muscular diseases, disorders,and conditions. In addition, reprogrammed cells may be used forreconstructive applications, such as for repairing or replacing tissuesor organs.

With respect to the therapeutic methods of the invention, it is notintended that the administration of RPSCs to a mammal be limited to aparticular mode of administration, dosage, or frequency of dosing; thepresent invention contemplates all modes of administration, includingintramuscular, intravenous, intraarticular, intralesional, subcutaneous,or any other route sufficient to provide a dose adequate to prevent ortreat a disease. The RPSCs may be administered to the mammal in a singledose or multiple doses. When multiple doses are administered, the dosesmay be separated from one another by, for example, one week, one month,one year, or ten years. One or more growth factors, hormones,interleukins, cytokines, or other cells may also be administered before,during, or after administration of the cells to further bias themtowards a particular cell type.

The RPSCs of the present invention may be used as an in vitro model ofdifferentiation, in particular for the study of genes which are involvedin the regulation of early development. Differentiated cell tissues andorgans using the RPSCs may be used in drug studies.

Furthermore, the RPSCs produced according to the invention maybeintroduced into animals, e.g., SCID mice, cows, pigs, e.g., under therenal capsule or intramuscularly and used to produce a teratoma therein.This teratoma can be used to derive different tissue types. Also, theinner cell mass produced by X-species nuclear transfer may be introducedtogether with a biodegradable, biocompatible polymer matrix thatprovides for the formation of 3-dimensional tissues. After tissueformation, the polymer degrades, ideally just leaving the donor tissue,e.g., cardiac, pancreatic, neural, lung, liver. In some instances, itmay be advantageous to include growth factors and proteins that promoteangiogenesis. Alternatively, the formation of tissues can be effectedtotally in vitro, with appropriate culture media and conditions, growthfactors, and biodegradable polymer matrices.

Applications of the Somatic Cell Reprogramming Methods and RPSCs inAnimals

The reprogramming methods disclosed herein may be used to generate RPSCsfor a variety of animal species. The RPSCs generated can be useful toproduce desired animals. Animals include, for example, avians andmammals as well as any animal that is an endangered species. Exemplarybirds include domesticated birds (e.g., quail, chickens, ducks, geese,turkeys, and guinea hens) as well as other birds such as birds of prey(e.g., hawks, falcons, ospreys, condors, etc.), endangered birds (e.g.,parrots, California condor, etc.), ostriches etc. Exemplary mammalsinclude murine, caprine, ovine, bovine, porcine, canine, feline andprimate. Of these, preferred members include domesticated animals,including, for examples, cattle, buffalo, pigs, horses, cows, rabbits,guinea pigs, sheep, and goats.

RPSCs generated by the reprogramming methods of the present inventionallows one, for the first time, to genetically engineer animals forwhich ES cells are not available through other means. RPSCs are ES-likecells, and are thus amenable to genetic manipulation. To date, no EScells are available for a wide variety of animals. As a result, forthese animals, it is currently practically impossible to creategenetically modified animals having targeted mutations. The ES-cell likeRPSCs can be manipulated to introduce desired targeted geneticmodifications. The resulting engineered RPSCs can then be used togenerate a cloned animal with the desired genetic modifications in itsgerm line, using methods described for ES cells in mouse. See Capecchiand Thomas, U.S. Pat. Nos. 5,487,992, 5,627,059, 5,631,153, and6,204,061. Genetic engineering in animals has potentially greatapplications in a variety of animals, especially farm animals.

The somatic cell reprogramming methods of the present invention providesat least two methods for delivering optimized farm animals. In thefirst, somatic cell reprogramming can be used to capture the bestavailable phenotype for a farm animal stock. The current technologiesused to deliver optimized farm animals are based on selective breeding,and expansion from preferred breeding stocks. Animals that have beenselected on the basis of superior characteristics, including, forexample, meat content, egg production (in the case of poultry), feedconversion ratio, are used to breed large numbers of animals that are inturn used in the human food supply. This traditional process hasprofound inherent inefficiencies. The phenotype observed in anindividual animal is often only partially transmitted in the progeny ofthat animal. Therefore, traditional breeding schemes are inefficient incapturing the very best phenotype in all of the progeny animals. Incontrast, the reprogramming methods of the present invention provides acontrolled and efficient way to achieve the same goal, by generatingRPSCs from somatic cells of an animal with the desired characteristics.The RPSCs generated may be used immediately to generate cloned animalsderived from the RPSCs. Known methods for generating mice from ES cellscan be used for this procedure. Alternatively, the RPSCs generated maybe cryopreserved and thawed in response to a grower's needs.

In the second method, somatic cells from an animal with the desiredcharacteristics are reprogrammed to produce RPSCs. The RPSCs are furthergenetically engineered to introduce desired genetic modification(s),before being placed into a recipient embryo to produce desired progeny.

The reprogramming methods can also be used to rescue endangered species.Somatic cell reprogramming provides an efficient method to generateRPSCs from somatic cells of an endangered animal. The resulting RPSCscan be used immediately to expand the numbers of the endangered animal.Alternatively, the RPSCs can be cryopreserved to generate a RPSC stockfor the endangered species, as a safeguard measure against extinction ofthe endangered species.

The subject invention will be more particularly described with referenceto the following non-limiting examples. All patents, patent applicationsand references cited herein are incorporated in their entirety byreference. In addition, the teachings of U.S. Provisional ApplicationNo. 60/525,612, filed Nov. 26, 2003, U.S. Provisional Application No.60/530,042, filed Dec. 15, 2003, U.S. Provisional Patent Application No.60/922,121, filed Apr. 7, 2007, and U.S. patent application Ser. No.10/997,146, filed Nov. 24, 2004, are incorporated herein by reference.

EXAMPLES Example 1

Methods

Cell Culture, MEF Isolation and Viral Infections

ES and iPS cells were cultivated on irradiated MEFs in DME containing15% fetal calf serum, Leukemia Inhibiting Factor (LIF),penicillin/streptomycin, L-glutamine, and non-essential amino acids. Allcells were depleted of feeder cells for two passages on 0.2% gelatinbefore RNA, DNA or protein isolation. Transgenic MEFs were isolated andselected in 2 μg/ml puromycin (Sigma) from E13.5 chimeric embryosfollowing blastocyst injection of Oct4-inducible KH2 ES cells(Hochedlinger et al., Cell 121(3):465 (2005)) which had been previouslytargeted with either Oct4-IRES-GfpNeo or Nanog-neo constructs (Mitsui etal., Cell 113(5):631 (2003)). 2×105 MEFs at passage 3-4 were infectedovernight with pooled viral supernatant generated by transfection ofHEK293T cells (Eugene, Roche) with the Moloney-based retroviral vectorpLIB (Clontech) containing the cDNAs of Oct4, Sox2, Klf4 and c-Myctogether with the packaging plasmid pCL-Eco (Naviaux et al., J Virol70(8):5701 (1996)).

Southern Blot, Methylation and Chromatin Analysis

To assess the levels of DNA methylation, genomic DNA was digested withHpaII, and hybridized to pMR150 as a probe for the minor satelliterepeats (Chapman et al., Nature 284 (1984)), or with an IAP-probe (Walshat al., Nat Genet 20(2):116 (1998)). Bisulfite treatment was performedwith the Qiagen EpiTect Kit. For the methylation status of Oct4 andNanog promoters bisulfite sequencing analysis was performed as describedpreviously (Blelloch et al., Stem Cells 24(9):2007 (2006)). 10-20 clonesof each sample were sequenced in both directions. For imprinted genes, aCOBRA assay was performed. PCR primers and conditions were as describedpreviously (Lucifero at al., Genomics 79(4):530 (2002)). PCR productswere gel purified, digested with BstUI or HpyCH4 IV and resolved on a 2%agarose gel. The status of bivalent domains was determined by chromatinimmunopreciptation followed by quantitative PCR analysis as describedbefore (Boyer at al., Nature 441:349 (2006)).Expression Analysis50 ng of total RNA isolated using TRIzol reagent (Invitrogen) wasreverse transcribed and quantified using QuantTtect SYBR green RT-PCRKit (Qiagen) on a 7000 ABI detection system. Western blot andimmunofluorescence analysis was performed as described (Hochedlinger etal., Cell 121(3):465 (2005); Wernig et al., J. Neurosci 24(22):5258(2004)). Primary antibodies included Oct4 (monoclonal mouse, SantaCruz), Nanog (polyclonal rabbit, Bethyl), actin (monoclonal mouse,Abcam), SSEA1 (monoclonal mouse, Developmental Studies Hybridoma Bank).Appropriately labeled secondary antibodies were purchased from JacksonImmunoresearch. Microarray targets from 2 μg total RNA were synthesizedand labeled using the Low RNA Input Linear Amp Kit (Agilent) andhybridized to Agilent whole mouse genome oligo arrays (G4122F). Arrayswere scanned on an Agilent G2565B scanner and signal intensities werecalculated in Agilent FE software. Datasets were normalized using a Rscript and clustered as previously described (Brambrink et al., ProcNatl Aced Sci USA 103(4):933 (2006)). Microarray datasets were submittedto the ArrayExpress database.ResultsOct4-Induced Fibroblasts are More Susceptible to Reprogramming thanUninduced Fibroblasts as Demonstrated by Nuclear Transfer ExperimentA. Generation of Transgenic Mouse Carrying an Inducible Oct4 Transgene

An inducible Oct4 allele was constructed as follows: first, twointegration vectors are constructed. The first integration vector,inducible Oct4 integration vector, contains an Oct4 gene driven by atetracycline-inducible promoter (Tet-Op). The Tet-Op-Oct4 cassette isflanked by a splice-acceptor double poly-A signal (SA-dpA) at its 5′ endand a SV40 polyA tail (SV40-pA) at its 3′ end. The second integrationvector, tetracycline activator integration vector, contains a mutantform of tetracycline activator, M2-rtTA, which is more responsive todoxycycline (Dox) induction than the wild type activator. (Urlinger etal., Proc Natl Acad Sci USA 97(14):7963 (2000)).

The two integration vectors are introduced into V6.5 ES cells: theinducible Oct4 integration vector and the tetracycline activatorintegration vector are introduced into the Collagen locus and the Rosa26locus respectively via site-specific integration, as shown in FIG. 1.The resulting ES cells are used to make Oct4-inducible mice bytetraploid complementation.

B. Expression of the Inducible Oct4 Transgene

Fibroblasts derived from tail biopsies of the Oct4-inducible mice werecultured. A fraction of the cultured fibroblasts were induced withdoxycycline for 3 days (at 2 microgram/ml), and Oct4 expression wasdetected by Northern blot and Western blot analysis. The Oct4 expressionlevel in fibroblasts treated with doxycycline is comparable to the Oct4expression level in ES cells, and undetectable in fibroblasts nottreated with doxycycline. The expression results demonstrate that theinducible Oct4 transgene is expressed as planned.

C. Nuclear Transfer Experiment

Nuclear transfer was performed on fibroblasts derived from tail biopsiesof mice that carry the inducible Oct4 transgene. Dox induction was for24 hours prior to nuclear transfer. Cloned embryos were then activatedand cultured to the blastocyst stage to derive ES cells as describedpreviously (Hochedlinger and Jaenisch, Nature 415:1035 (2002)). Onaverage, blastocyst formation and ES cell derivation (as measured as afraction of eggs with pronucleus formation) is more efficient fromOct4-induced fibroblast than from uninduced fibroblasts. This resultdemonstrated that induced Oct4 expression in somatic cells such asfibroblasts make these cells more susceptible to reprogramming.

Selection of ES-Like Cells by Stringent Criteria

Using homologous recombination in ES cells, we generated mouse embryonicfibroblasts (MEFs) that carried a neomycin resistance marker insertedinto either the endogenous Oct4 (Oct4-neo) or Nanog locus (Nanog-neo)(FIG. 2A). These cultures were sensitive to G418, indicating that theOct4 and Nanog loci were, as expected, silenced in somatic cells. Fivedays after infection with Oct4-, Sox2-, c-Myc- and Klf4-expressingretroviral vectors the cells were passaged, and G418 was added to thecultures to select for drug resistant cells. Resistant colonies appearedin both the Nanog-neo and the Oct4-neo cultures, though with a verydifferent efficiency: the number of drug resistant colonies in theNanog-neo cultures was 35 fold higher than in the Oct4-neo cultures(FIG. 2B). When the colonies were stained for alkaline phosphatase (AP)or SSEA1, a significantly higher fraction of the Oct4-neo colonies waspositive and showed an ES cell like morphology. This suggests thatalthough the Nanog locus was easier to activate, a higher fraction ofthe drug resistant colonies in Oct4-neo cultures were reprogrammed to apluripotent state. Consistent with this notion, out of 12 randomlypicked Oct4-neo colonies, ten continued to proliferate and maintain anES-like phenotype, and three of these displayed strong AP activity andSSEA1 expression. In contrast, all nine continuously proliferatingNanog-neo colonies had a flat or small and round-shaped appearance, andthe rare ES cell-resembling colonies were only partially labeled withSSEA1 antibodies. However, after careful morphological selection ofcolonies from both selection strategies based on criteria known in theart to be characteristic of ES cells, we were able to propagate ES-likeclones (designated as iPS cells for “induced pluripotent cells”) whichdisplayed homogenous Nanog, SSEA1 and AP expression and formedundifferentiated colonies when seeded at clonal density ongelatin-coated dishes.

Characterization of Gene Expression and DNA Methylation in iPS Cells

To characterize the reprogrammed cells on a molecular level, we usedquantitative RT-PCR (qRT-PCR) to measure expression of ES cell andfibroblast-specific genes. Oct4-neo-selected iPS cells expressedendogenous Nanog and Oct4 at similar levels as ES cells, whereas MEFsdid not express either gene. Using specific primers to distinguishendogenous from viral Sox2 transcripts showed that the vast majority ofSox2 transcripts originated from the endogenous locus. In contrast,HoxA9 and Zfpm2 were highly expressed in MEFs but at very low levels iniPS or ES cells. Western analysis showed similar Nanog and Oct4 proteinlevels in iPS and ES cells. Finally, we used microarray technology tocompare gene expression patterns on a global level. The iPS cellsclustered with ES cells in contrast to wild type or donor MEFs.

To investigate the DNA methylation level of Oct4 and Nanog promoters, weperformed bisulfite sequencing and COBRA analysis with DNA isolated fromES cells, iPS cells and MEFs. Both loci were demethylated in ES and iPScells and fully methylated in MEFs. To assess whether the maintenance ofgenomic imprinting was compromised, we assessed the methylation statusof four imprinted genes H19, Peg1, Peg3 and Snrpn. Bands correspondingto an unmethylated and methylated allele were detected for each gene inMEFs, iPS cells and tail tip fibroblasts. In contrast, EG cells, whichhave erased all imprints (Labosky et al., Development 120(11):3197(1994)), were unmethylated. Our results indicate that the epigeneticstate of the Oct4 and Nanog genes was reprogrammed from atranscriptionally repressed (somatic) state to an active (embryonic)state and that the pattern of somatic imprinting was maintained in iPScells.

Recently, downstream target genes of Oct4, Nanog and Sox2 have beendefined in ES cells by genome wide location analyses (Boyer et al., NatGenet 38(4):431 (2006)). These targets include many importantdevelopmental regulators, a proportion of which are also bound andrepressed by the PcG complexes PRC1 and PRC2 (Lee et al., Cell125(2):301 (2006); Boyer et al., Nature 441(7091):349 (2006)). Notably,the chromatin at many of these non-expressed target genes adopt abivalent conformation in ES cells, carrying both the “active” histone H3lysine 4 (H3K4) methylation mark and the “repressive” histone H3 lysine27 (H3K27) methylation mark (Bernstein et al., Cell 125(2): 315 (2006);Azuara et al., Nat Cell Biol 8(5):532 (2006)). In differentiated cells,those genes tend to instead carry either H3K4 or H3K27 methylation marksdepending on their expression state.

We used chromatin immunoprecipitation (ChIP) and real-time PCR toquantify H3K4 and H3K27 methylation for a set of genes reported to bebivalent in pluripotent ES cells (Bernstein et al., Cell 125(2): 315(2006)). In the MEFs, the expressed genes Zfpm2 and HoxA9 carry strongH3K4 methylation, but weaker or no H3K27 methylation, whereas Nkx2.2,Sox1, Lbx1h, Pax5 and Evx1 predominantly carry H3K27 methylation. Whenanalyzing Oct4-neo iPS cells, however, we found at each of these genes abivalent conformation with both histone modifications like in normal EScells (Bernstein et al., Cell 125(2): 315 (2006)). Identical resultswere obtained in several iPS clones selected from Oct4-neo and Nanog-neofibroblasts.

iPS Cells are Resistant to Global Demethylation

Tolerance of genomic demethylation is a unique property of ES cells assomatic cells undergo rapid apoptosis upon loss of themethyltransferase. We investigated whether iPS cells would be resistantto global demethylation after Dnmt1 inhibition and would be able tore-establish global methylation patterns after restoration of Dnmt1activity. To this end, we utilized a conditional lentiviral vectorcontaining a Dnmt1 targeting shRNA and a GFP reporter gene (Ventura atal., Proc Natl Acad Sci USA 101(28):10380 (2004)). Infected iPS cellswere plated at low density and GFP-positive colonies were picked andexpanded. Southern analysis using HpaII digested genomic DNA showed thatglobal demethylation of infected iPS cells was similar to Dnmt1−/− ES incontrast to uninfected iPS cells or MEFs, which displayed normalmethylation levels.

Morphologically, the GFP-positive cells were indistinguishable from theparental line or from uninfected sister subclones indicating that iPScells tolerate global DNA demethylation. In a second step, the Dnmt1shRNA was excised through Cre-mediated recombination and normal DNAmethylation levels were restored as has been reported previously for EScells (Holm et al., Cancer Cell 8(4):275 (2005)). These observationsshow the functional reactivation of the de novo methyltransferasesDnmt3a/b in iPS cells (Okano at al., Cell 99:247 (1999)). As expected,the imprinted genes Snrpn and Peg3 were unmethylated and resistant toremethylation.

Retroviral Vectors are Silenced by De Novo Methylation in iPS Cells

Southern analysis indicated that the Oct4-neo iPS clone 18 carried 4-6copies of the Oct4, c-Myc and Klf4 and only 1 copy of Sox2 retroviralvectors. Because these four factors were under the control of theconstitutively expressed retroviral LTR, it was unclear in a prior studywhy iPS cells could be induced to differentiate (Takahashi and Yamanaka,Cell 126 (4):663 (2006)). To address this question, we designed primersspecific for the 4 viral-encoded factor transcripts and comparedexpression levels by qRT-PCR in MEFs 2 days after infection, in iPScells, in embryoid bodies (EB) derived from iPS cells and indemethylated and remethylated iPS cells. Although the MEFs represented aheterogenous population composed of uninfected and infected cells, viraldependent Oct4, Sox2, c-Myc and Klf4 RNA levels were 5-fold lower in iPScells than in the infected MEFs, suggesting silencing of the viral LTRby de novo methylation upon reprogramming of the MEFs. Consistent withthis conclusion is the fact that the total Sox2 and Oct4 RNA levels iniPS cells was similar to that in wild type (wt) ES cells and that theSox2 transcripts in iPS cells were mostly, if not exclusively,transcribed from the endogenous gene. Upon differentiation to EBs, bothviral and endogenous transcripts were downregulated. Importantly, allviral Sox2, Oct4 and Klf4 transcripts were about 2-fold upregulated inDnmt1 knock down iPS cells and again downregulated following restorationof Dnmt1 activity. In contrast, transcript levels of c-Myc were about20-fold lower in iPS cells than in infected MEFs and did not change upondifferentiation of demethylation. Our results suggest that theretroviral vectors are subject to silencing by de novo methylation uponreprogramming of the fibroblasts.

iPS Cells have Similar Developmental Potential as ES Cells

We determined the developmental potential of iPS cells by teratoma andchimera formation. Histological analysis of tumors formed 3 weeksfollowing subcutaneous injection of iPS cells into SCID mice revealedthat the cells had differentiated into various cell types representingall three embryonic germ layers. Importantly, Oct4 and Nanog were onlyexpressed in cells that appeared undifferentiated but were silenced indifferentiated cells as in teratomas resulting from the injection of wtES cells. To more stringently assess the developmental potential of iPScells, GFP-labeled subclones were injected into diploid (2N) ortetraploid (4N) blastocysts. Injection of cells into 4N blastocysts isthe most rigorous test for developmental potency, as the resultingembryo is composed only of the injected donor cells (“all ES embryo”).iPS cells derived from Oct4-neo and Nanog-neo MEFs could generate “alliPS embryos.” Injection of iPS cells into 2N blastocysts efficientlygenerated high-contribution prenatal and viable postnatal chimeras.These findings indicate that iPS cells can contribute to all lineages ofthe embryo and thus have a similar developmental potential as ES cells.

The results presented in Example 1 confirm that the four transcriptionfactors Oct4, Sox2, c-Myc and Klf4 can induce epigenetic reprogrammingof a somatic genome to an embryonic state though with low efficiency.These four factors were initially identified based on their ability toinduce expression of the Fbx15 gene in somatic cells. Fbx15 isspecifically expressed in mouse ES cells and early embryos but isdispensable for maintenance of pluripotency and mouse development(Takahashi and Yamanaka, Cell 126(4):663 (2006)). In contrast to cellsselected based on their expression of Fbx15, fibroblasts that hadreactivated the endogenous Oct4 (Oct4-neo) or Nanog (Nanog-neo) locigrew feeder independently, expressed normal Oct4, Nanog and Sox2 RNA andprotein levels, were epigenetically identical to ES cells by a number ofcriteria and were able to generate viable chimeras. Transduction of the4 factors generated 35-fold more drug resistant cells from Nanog-neothan from Oct4-neo fibroblasts but a higher fraction of Oct4-selectedcells exhibited all characteristics of pluripotent ES cells that wereassessed.

The data presented above suggests that the pluripotent state of iPScells is induced by the virally-transduced factors but is largelymaintained by the activity of the endogenous pluripotency factorsincluding Oct4, Nanog and Sox2 because the viral controlled transcripts,though expressed highly in MEFs, become mostly silenced in iPS cells.The total levels of Oct4, Nanog and Sox2 were similar in iPS and wt EScells. Consistent with the conclusion that the pluripotent state ismaintained by the endogenous pluripotency genes is the fact that theOct4 and the Nanog genes become hypomethylated in IFS as in ES, and thatthe bivalent histone modifications of developmental regulators wasreestablished. Importantly, iPS cells were resistant to globaldemethylation induced by inactivation of Dnmt1 similar to ES cells andin contrast to somatic cells. Re-expression of Dnmt1 in thehypomethylated ES cells resulted in global remethylation indicating thatthe iPS cells had also reactivated the de novo methyltransferasesDnmt3a/b. All these observations are consistent with the conclusion thatthe iPS cells have gained an epigenetic state that is similar to that ofnormal ES cells.

Expression of the 4 factors proved to be a robust method to inducereprogramming of somatic cells to a pluripotent state. One object of thepresent invention is to provide new ways to identify small moleculesthat reprogram cells without gene transfer of potentially harmfulgenetic material.

Example 2 Methods

Cell Culture, MEF Isolation and Viral Infections

ES and iPS cells were cultivated on irradiated MEFs in DME containing15% fetal calf serum, Leukemia Inhibiting Factor (LIF),penicillin/streptomycin, L-glutamine, beta-mercaptoethanol andnon-essential amino acids. All cells were depleted of feeder cells fortwo passages on 0.2% gelatin before RNA, DNA or protein isolation. 2×10⁵MEFs at passage 3-4 were infected overnight with pooled viralsupernatant generated by transfection of HEK293T cells (Fugene, Roche)with the Moloney-based retroviral vector pLIB (Clontech) containing thecDNAs of Oct4, Sox2, Klf4 and c-Myc together with the packaging plasmidpCL-Eco (Naviaux et al., J Virol 70:5701 (1996)).

Blastocyst Injection

Diploid or tetraploid blastocysts (94-98 hours post HCG injection) wereplaced in a drop of DMEM with 15% PCS under mineral oil. A flat tipmicroinjection pipette with an internal diameter of 12-15 mm was usedfor ES cell injection. A controlled number of ES cells were injectedinto the blastocyst cavity. After injection, blastocysts were returnedto KSOM media and placed at 37° C. until transferred to recipientfemales.

Recipient Females and Caesarean Sections

Ten to fifteen injected blastocysts were transferred to each uterinehorn of 2.5 days postcoitum pseudopregnant B6D2F1 females. To recoverfull-term ES or chimeric pups, recipient mothers were sacrificed at 19.5days postcoitum. Surviving pups were fostered to lactating BALE/cmothers.

Viral Integrations

Genomic DNA was digested with SpeI overnight, followed byelectrophoresis and transfer. The blots were hybridized to therespective radioactively labeled cDNAS.

Immunohistochemistry

Cells were fixed in 4% paraformaldehyde for 10 min at room temperature,washed 3 times with PBS and blocked for 15 min with 5% FBS in PBScontaining 0.1% Triton. After incubation with primary antibodies againstSox2 (monoclonal mouse, R&D Systems), Oct4 (monoclonal mouse, SantaCruz), c-myc (polyclonal rabbit, Upstate), Nanog (polyclonal rabbit,Bethyl) and SSEA1 (monoclonal mouse, Developmental Studies HybridomaBank) for 1 hour cells were washed 3 times with PBS and incubated withfluorophore-labeled appropriate secondary antibodies purchased fromJackson Immunoresearch. Specimen were analyzed on an OlympusFluorescence microscope and images were acquired with a Zeiss Axiocamcamera.

Results

Reprogramming of Somatic Cells without Genetic or Chemical Selection

As described above, in vitro reprogramming of somatic cells into apluripotent ES cell-like state has been achieved through retroviraltransduction of Oct4, Sox2, c-myc and Klf4 into murine fibroblasts. Inthese experiments the rare “induced Pluripotent Stem” (iPS) cells wereisolated by stringent selection for activation of a neomycin resistancegene inserted into the endogenous Oct4 or Nanog loci. Direct isolationof pluripotent cells from cultured somatic cells is of potentialtherapeutic interest but in order to translate such methods tonon-murine, e.g., human, systems it would be desirable to developalternatives to the requirement for transgenic donors used in the iPSisolation protocol described above. Here we demonstrate for the firsttime that reprogrammed pluripotent cells can be isolated fromgenetically unmodified somatic donor cells solely based uponmorphological criteria. Thus, for example, genetically unmodifiedsomatic donor cells can be obtained from a mouse, a rat, a rabbit, afarm animal, a companion animal, a primate or a human, and reprogrammedpluripotent cells can be derived from these donor cells.

Somatic cell nuclear transfer and cell fusion with embryonic stem (ES)cells have been well-established approaches to achieve reprogramming ofsomatic nuclei into a pluripotent state. Direct in vitro isolation ofpluripotent ES-like cells from cultured somatic cells was achievedrecently by transduction of the four transcription factors Oct4, Sox2,Klf4 and c-myc (below referred to as “factors”) into geneticallymodified fibroblasts. The selection for the rare reprogrammed inducedPluripotent Stem (iPS) cells was based upon the reactivation of theFbx15 (Takahashi and Yamanaka, Cell 226:663 (2006)) or the Oct4 or Nanoggenes, all of which carried a drug resistance marker inserted into therespective endogenous loci by homologous recombination or a transgenecontaining the Nanog promoter. While iPS cell isolation based upon Fbx15activation yielded cells that were pluripotent, they differed from EScells at the molecular level and were unable to generate live chimeras.In these experiments selection was initiated at 3 days after viraltransduction. In contrast, selection for Oct4 or Nanog activationproduced pluripotent iPS cells that were epigenetically and biologicallyindistinguishable from normal ES cells. Reprogramming to pluripotencywas, however, a slow and gradual process involving the sequentialactivation of the ES cell markers alkaline phosphatase (AP), SSEA1 andNanog over a period of 2-4 weeks after factor transduction. Thus, whenG418 was added to cultures of Oct4-neo or Nanog-neo fibroblasts at 3days after factor transduction, no drug resistant colonies were formed,whereas addition of drug at 1 week generated a few and addition at 2weeks significantly more drug resistant and reprogrammed colonies. Theinverse relationship between the time of drug selection after factortransduction and the number of drug resistant iPS cells is consistentwith the notion that the process of reprogramming involves multiplestochastic events that convert the epigenetic state of a somatic to thatof a pluripotent cell.

In the present Example we show that pluripotent iPS cells can be derivedfrom normal, genetically unmodified donor cells. In the first set ofexperiments we used a GFP marker inserted into the Oct4 locus to monitorthe reprogramming process. Mouse embryonic fibroblasts (MEFs) carryingan IRES-EGFP cassette in the Oct4 locus were transduced with the fourfactors Oct4, Sox2, c-myc and Klf4 by retrovirus-mediated gene transferas described before. Three days after infection the fibroblasts becamemorphologically more diverse than uninfected control cells and foci ofincreased growth appeared. On day 6, small tightly packed andsharp-edged colonies developed resembling ES cell colonies. During thefollowing days these colonies continued to grow into large and moreheterogeneous cell aggregates with some sectors resembling ES cell-likegrowth while more small and tight colonies continued to appear.

Eight of these large colonies were picked on day 11 and ten additionalcolonies were picked on day 16 based solely upon their morphology. Whenexamined under the fluorescence microscope no GFP expression wasdetectable at day 11 and only one of the ten colonies picked on day 16showed weak GFP expression. One of the eight colonies picked on day 11and four of the ten colonies picked on day 16 gave rise to homogenous,ES-like cell lines. All five lines initiated Oct4-EGFP expression within1-3 passages (Table 1) and displayed homogenous AP activity as well asSSEA1 and Nanog expression as would be expected for fully reprogrammediPS cells.

Of the remaining colonies that had been picked initially based onmorphological criteria, ten gave rise to heterogeneous culturescontaining mainly fibroblast-like cells interspersed with a few ES-likecolonies (Table 1). We investigated whether these heterogeneous cultureswould yield additional iPS cell lines upon further passaging. For thiswe picked three ES-like colonies from each of five mixed culturesderived from the initial outgrowths and successfully established fiveadditional iPS cell lines within 2-3 passages (Tables 1 and 2). In orderto test whether the observed heterogeneity was a result of partlyincomplete reprogramming or a contamination of not reprogrammedfibroblasts, we FACS-sorted the GFP positive and negative cells fromclone #5 and the heterogenous subclone #5.2 and compared proviralintegration patterns using southern blot analysis. The results indicatedthat the two cell populations are derived from the same parental cellindicating the requirement of further epigentic events. From the pickedsubclones that did not generate secondary iPS lines, three subclones(6.1, 6.2 and 6.3; see Table 1 and 2) displayed an altered morphology(small cells, tightly grown colonies) but remained Oct4-GFP negativeover multiple passages and displayed no staining for AP, SSEA1 or Nanog,suggesting that these cells were not pluripotent. The occurrence of ESmarker negative cells was rare and these cells displayed subtlemorphological differences from ES or true iPS cells such as the shape ofcolony boundaries. Because the cells were infected with all fourretroviruses, it is possible that the four factors may not have beenexpressed at the right levels, giving rise to transformed rather thanpluripotent cells. For example, high c-myc/Klf4 and insufficientOct4/Sox2 expression may lead to rapidly growing non-iPS cellsconsistent with the notion that the role of Oct4 and Sox2 in thereprogramming process may be the suppression of the c-myc and Klf4transformed phenotype (Yamanaka, Stem Cells 1:39-49 (2007)).

All iPS cell lines tested showed GFP intensity comparable to theOct4-GFP ES cells consistent with our previous observation that Oct4protein levels were similar in different iPS cell lines (Wernig, 2007).To analyze whether the iPS cells isolated by morphological criteriaremained phenotypically stable over time, GFP fluorescence was monitoredafter multiple passages. These results show that the iPS cells exhibitednon-variable and robust Oct4-GFP expression up to at least ninepassages. These data clearly demonstrate that stable iPS lines can beefficiently derived without relying on drug selection.

We used the fraction of virus infected input cells and the number of EScell-like colonies to estimate the efficiency of reprogramming. In atypical experiment about 100,000 cells were exposed to virus. Usingstaining for Sox2, Oct4 and c-myc as criterion we estimated about 10.2%of the cells were infected with all four virus generating 115 EScell-like colonies. The efficiency for deriving iPS cells from thenumber of picked colonies was 44%. Thus, the overall efficiency ofreprogramming was extrapolated to be about 0.5%.

Finally, we evaluated the developmental potency of non-selected iPScells by teratoma formation, and injections into diploid (2N) andtetraploid (4N) blastocysts (Table 3) (Eggan at al., Proc Natl Acad SciUSA 98:6209-6214 (2001)). Three weeks after subcutaneous injection intoSCID mice, lines 8.1 and 14 developed tumors which contained tissuetypes from all three germ layers determined by histological analysis.Following injection into 2N blastocysts, we generated live postnatalanimals with high coat color chimerism. Importantly, when injected into4N blastocysts, which is the most stringent test for developmentalpotency, live E14.5 embryos could be recovered (Table 4). These datademonstrate that screening for iPS cells based upon morphologicalcriteria rather than selection for drug resistance can generatepluripotent iPS cells that display a similar biological potency as EScells.

Derivation of iPS Cells from Genetically Unmodified Donor Cells

In the experiments described above, the Oct4-GFP marker was used tomonitor the reprogramming process but not to screen for reprogrammed iPScells. To assess whether iPS cells can be derived from geneticallyunmodified donor cells, we generated wild type MEFs from Balb/c and129SvJae/C57B16(F1) mice and adult tailtip fibroblasts from129SvJae/C57B16 (F1) and C57B16/DBA (F1) 2-3 month old mice. The cellswere infected with retroviruses encoding the four factors and largecolonies were picked at day 16 or later as described above. As in theprevious experiments ES-like colonies became visible within one passageafter picking of the primary colonies. Upon continued passaging orthrough subcloning we readily established homogenous cell lines with EScell morphology and growth properties.

Assuming that reprogrammed cells outgrow the donor fibroblasts, weattempted to generate iPS cells by passaging the entire plate instead ofpicking colonies following morphological criteria. Many small coloniesperfectly resembling ES cell colonies appeared within several days afterthe first passage of infected cell populations and 5 out 6 pickedcolonies grew into stable iPS lines (Table 3), After 2-3 passages usingeither direct picking or passaging the whole plate followed by pickingof individual colonies we established one or more iPS lines from eachbackground (Table 3). All genetically unmodified iPS lines expressed AP,SSEA1 and Nanog. In addition we generated chimeric embryos from Balb/cand 129/B6 MEF derived iPS lines, demonstrating that iPS cells fromgenetically unmodified fibroblasts are pluripotent (Table 4). It shouldbe noted, however, that passaging of the factor transduced cellpopulations, while representing a simplified isolation protocol, cannotexclude that individual iPS cell lines may have been derived from thesame reprogrammed parental cell.

Our results suggest that in vitro reprogramming of fibroblasts occursfrequently enough be detected in cultures of non-transgenic donor cellsand is stable without selective pressure to express Oct4 or Nanog. Thus,the four factor-induced reprogramming can be applied to wild type cells.Without being bound by any theory, it appears that ectopic expression ofOct4, Sox2, c-myc and Klf4 initiates a gradual reprogramming process inmultiple infected cells that ultimately leads to pluripotency over atime period of several weeks. Using Oct4 GFP MEFs to monitorreactivation of the endogenous Oct4 locus we found that all colonies butone were GFP-negative at the time of picking (see Table 2) and becameGFP positive only after several passages. This suggests thatreprogramming is a slow process involving the sequential activation ofES cell markers such as AP, SSEA1 and Nanog with Oct4 activationrepresenting one of the last epigenetic events in the process. Also,these observations are consistent with our previous finding that thenumbers of reprogrammed colonies were lower when drug selection for Oct4activation was applied early after viral transduction, but wassignificantly higher when drug selection was initiated later. Finally,the slow reprogramming process induced by factor transduction mayexplain why the drug selection for Fbx15 activation as early as 3 daysafter infection as used in the initial iPS isolation protocol yieldedonly cells that had undergone incomplete epigenetic reprogramming. Ourresults predict that selection for Fbx15 activation at later times wouldgenerate iPS cells that are similar to iPS cells selected for Oct4activation or isolated based on morphological criteria.

Example 3 Methods

Cell Culture and Viral Infections.

ES and established iPS cells were cultured on irradiated MEFs in DMEcontaining 15% FCS, leukemia inhibiting factor (LIF),penicillin/streptomycin, L-glutamine, beta-mercaptoethanol andnonessential amino acids. MEFs used to derive primary iPS lines byinfections with inducible lentiviruses were harvested at 13.5dpc from F1matings between ROSA26-M2rtTA mice (Beard et al., 2006) and Nanog-GFPmice (Brambrink et al., 2008). Mouse C/EBPα cDNA was cloned into EcoRIcloning site of pLib, MSCV-Neo and pMig retroviral vectors. pMXs vectorsencoding ES pluripotency genes were previously described (Takahashi andYamanaka, 2006). Lentiviral preparation and infection withDoxycycline-inducible lentiviruses encoding Oct4, Klf4, c-Myc and Sox2cDNA driven by the TetO/CMV promoter were previously described(Brambrink, 2008). Retrovirus stocks were prepared by transienttransfection of Phoenix-Eco cells using Fugene (Roche), and supernatantswere harvested 48 hr later. For infection, purified B cell subsets wereresuspended in IMDM with 15% FCS as well as IL-4, IL-7, Flt-3L, SCF (10ng/ml each, Peprotech), anti-CD40 (0.1 μg/ml, BD-Biosciences), BPS (10ng/ml, Sigma-Aldrich) and Dox (4 μg/ml). Then, 2 ml aliquots were platedonto a 24-well plate precoated with retronectin (Takara) followed by 2ml of retrovirus supernatant to which polybrene (Sigma) was added (8μg/ml). The plates were incubated at 37° C. for 2 hours, and afterward 1ml of viral supernatant was replaced with B cells resuspended in thecytokine-conditioned media described above. Plates were centrifuged for90 min at 900 RPM and than incubated 24 hours at 37° C. 5% CO₂. Infectedcells were then transferred onto OP9 bone marrow stromal cells line(ATCC) in fresh cytokine and Dox-supplemented media. After 14 days onDox, colonies were picked and cultured on MEF feeder cells in ES media(without hematopoietic cytokines or Dox) and in the presence ofpuromycin (2 μg/ml) to eliminate any remaining OP9 cells.

V(D)J Rearrangement Analysis.

IgH, Igκ and Igλ rearrangements were amplified by PCR using degenerateprimer sets as previously described (Chang et al., 1992; Cobaleda etal., 2007a; Schlissel at al., 1991) (Table 2). To characterizeindividual V-DJ rearrangements, the PCR fragments were cloned in TOPOvector, and at least 5 clones corresponding to the same PCR fragmentwere sequenced. Obtained sequences were analyzed with DNAPLOT searchengine (found at www.dnaplot.de). V-DJ and D-J rearrangements at the Ighlocus were detected by Southern blot analysis on genomic DNA of theindicated iPS lines digested with EcoRI and using a 3′JH4 probe (1.6-kbHindIII-EcoRI fragment of plasmid JH4.3) (Alt et al., 1981). Vκ-Jκrearrangements at the Igk locus were determined by Southern blotanalysis of BamHI-digested genomic DNA using a 3′Jκ5 probe (1-kbXbaI-EcoRV fragment of plasmid pBS-JκMAR) (Lewis et al., 1982).

DNA Methylation and Histone Marks Analysis.

For the methylation status of Oct4 and Nanog promoters, bisulphitesequencing analysis was performed as described previously (Wernig etal., 2007). A total of 10-20 clones of each sample was sequenced in bothdirections. The status of H3K4 and H3K27 bivalent domains was determinedby chromatin immunopercipitation followed by quantitative PCR analysis,as previously described (Bernstein et al., 2006).

Blastocyst Injections and Teratoma Formation.

Diploid or tetraploid blastocysts (94-98 h after HCG injection) wereplaced in a drop of DMEM with 15% FCS under mineral oil. A flat-tipmicroinjection pipette with an internal diameter of 12-15 mm was usedfor iPS cell injection (using a Piezo micromanipulator 34). A controllednumber of cells was injected into the blastocyst cavity. Afterinjection, blastocysts were returned to KSOM media and placed at 37° C.until transferred to recipient females. Ten to fifteen injectedblastocysts were transferred to each uterine horn of 2.5 days postcoitum pseudo-pregnant B6D2F1 females. To recover full-term pups,recipient mothers were killed at 19.5 days post coitum. Surviving pupswere fostered to lactating BALB/c mothers. For teratoma generation,2*10^(^6) cells were injected subcutaneously into both flanks ofrecipient SCID mice, and tumors were harvested for sectioning 3-6 weeksafter initial injection.

Immunofluorescence Staining.

Cells were fixed in 4% paraformaldehyde for 20 minutes at 25° C., washed3 times with PBS and blocked for 15 min with 5% FBS in PBS containing0.1% Triton-X. After incubation with primary antibodies against Nanog(polyclonal rabbit, Bethyl) and SSEA1 (monoclonal mouse, DevelopmentalStudies Hybridoma Bank) for 1 h in 1% FBS in PBS containing 0.1%Triton-X, cells were washed 3 times with PBS and incubated withfluorophore-labeled appropriate secondary antibodies purchased fromJackson Immunoresearch. Specimens were analyzed on an OlympusFluorescence microscope and images were acquired with a Zeiss Axiocamcamera.

Quantitative RT-PCR.

Bone marrow B cells were grown on OP9 cells in media supplemented withIL-7, SCF, Flt3, while spleen B cells were grown with IL-4, anti-CD40and LPS. OP9 cells were depleted by pre-plating on gelatin-coated platesbefore the cells were harvested for mRNA preparation. Puromycin wasadded to fibroblast (2 μg/ml) and B cell (0.3 μg/ml) cultures toeliminate non-transgenic cells. Total RNA was isolated using Rneasy Kit(Qiagen). Three micrograms of total RNA was treated with DNase I toremove potential contamination of genomic DNA using a DNA Free RNA kit(Zymo Research, Orange, Calif.). One microgram of DNase I-treated RNAwas reverse transcribed using a First Strand Synthesis kit (Invitrogen)and ultimately resuspended in 100 ul of water. Quantitative PCR analysiswas performed in triplicate using 1/50 of the reverse transcriptionreaction in an ABI Prism 7000 (Applied Biosystems, Foster City, Calif.)with Platinum SYBR green qPCR SuperMix-UDG with ROX (Invitrogen).Primers used for amplification were as follows: c-Myc: F,5′-ACCTAACTCGAGGAGGAGCTGG-3′ (SEQ ID NO: 1) and R,5′-TCCACATAGCGTAAAAGGAGC-3′ (SEQ ID NO: 2); Klf4: F,5′-ACACTGTCTTCCCACGAGGG-3′ (SEQ ID NO: 3) and R,5′-GGCATTAAAGCAGCGTATCCA-3′ (SEQ ID NO: 4); Sox2: F,5′-CATTAACGGCACACTGCCC-3′ (SEQ ID NO: 5) and R,5′-GGCATTAAAGCAGCGTATCCA-3′ (SEQ ID NO: 6); Oct4: F,5′-AGCCTGGCCTGTCTGTCACTC-3′ (SEQ ID NO: 7) and R,5′-GGCATTAAAGCAGCGTATCCA-3′ (SEQ ID NO: 8). To ensure equal loading ofcDNA into RT reactions, GAPDH mRNA was amplified using the followingprimers: F, 5′-TTCACCACCATGGAGAAGGC-3′ (SEQ ID NO: 9); and R,5′-CCCTTTTGGCTCCACCCT-3′ (SEQ ID NO: 10). Data were extracted from thelinear range of amplification. All graphs of qRT-PCR data shownrepresent samples of RNA that were DNase treated, reverse transcribed,and amplified in parallel to avoid variation inherent in theseprocedures. Gene expression analysis for ES markers was performed by PCRusing previously published primers (Takahashi and Yamanaka, 2006).

Flow Cytometry Analysis and Cell Sorting.

The following fluorescently conjugated antibodies (PE, FITC, Cy-Chromeor APC labeled) were used for FACS analysis and cell sorting: anti-SSEA1(RnD systems), anti-Igκ, anti-Igλ1, 2, 3, anti-CD19, anti-B220,anti-c-Kit, anti-CD25, anti-sIgM, anti-sIgD (all obtained fromBD-Biosciences). Cell sorting was performed by using FACS-Aria(BD-Biosciences), and consistently achieved cell sorting purity of >97%.For isolation of mature IgM+IgD+ B cells from spleen and lymph nodes,cells were depleted of Lin+ non-B cells by MACS sorting after stainingwith lineage markers antibodies (CD3e, CD4, CD8, CD11c, Gr1, c-Kit, Mac1and Ter119) prior to sorting.

Results

Inducible Expression of Reprogramming Factors in the B Cell Lineage

Initially work described herein sought to determine whether Oct4, Sox2,Klf4 and c-Myc transcription factors, which were shown to be sufficientto reprogram mouse and human fibroblast cultures (Meissner at al., 2007;Okita at al., 2007; Takahashi et al., 2007; Takahashi and Yamanaka,2006; Wernig at al., 2007) and mouse liver- and stomach-derived cellcultures (Aoi at al., 2008), were capable of reprogramming cells of theB cell lineage. Because of the relatively low infectivity of mouselymphocytes with viruses, we established a system that allowed inducibletransgenic expression of the four reprogramming factors in B cells.

To this end, we have recently shown that doxycycline-inducible (Dox)lentiviral vectors encoding the Oct4, Sox2, c-Myc and Klf4 transcriptionfactors are able to reprogram mouse embryonic fibroblasts (MEFs) intostable iPS cells that maintain their pluripotency after Dox withdrawal(Brambrink et al., 2008). When injected into blastocysts these cellswere capable of generating postnatal chimeras which contain clonalpopulations of somatic cells carrying the identical proviral copies thatgenerated the “primary” iPS cells (Brambrink et al., 2008). We reasonedthat B cells derived from these chimeras, when exposed to Dox underappropriate culture conditions, might activate the proviral copies thatinduced the primary iPS cells and thus might facilitate reprogrammingand the generation of “secondary” iPS cells (FIG. 3).

MEFs carrying a constitutively expressed reverse tetracyclinetrans-activator driven by the ROSA26 promoter (R26-M2rtTA) and aknock-in of GFP into the endogenous Nanog locus (Nanog-GFP) wereinfected with the Dox-inducible lentiviral vectors encoding Oct4, Sox2,c-Myc and Klf4 genes (Brambrink et al., 2008). Large macroscopiccolonies appearing after 12 days of Dox treatment were picked andpropagated without Dox to establish Nanog-GFP+ iPS lines, whichexpressed pluripotency markers alkaline phosphatase (AP), SSEA1 antigenand Oct4. The MEF-derived primary iPS cells were injected intoblastocysts to generate embryonic and adult chimeras. Pro-B(B220+c-Kit+) and Pre-B cells (B220+CD25+) (Cobaleda et al., 2007a) wereisolated from the bone marrow, and mature IgM+IgD+ B cells were purifiedfrom the spleen of 8 week old adult chimeric mice and grown in mediasupplemented with hematopoietic cytokines and Dox for 7 days. Afunctional puromycin resistance gene had been inserted into the ROSA26locus as part of the targeting strategy of M2rtTA (Brambrink et al.,2008) and allowed elimination of host-derived non-transgenic B cells bypuromycin selection (0.3 μg/ml).

Chimeras derived from MEF-iPS-#1 cell line were chosen for furtherstudy, as donor B cells from chimeras induced high expression levels ofthe 4 factors in the presence of Dox. Adult tail tip fibroblasts derivedfrom MEF-iPS #1 line also yielded Nanog-GFP+iPS lines following additionof Dox, though the expression levels of the four factors followingaddition of Dox were lower than those observed in B cells derived fromthe same chimera.

Reprogramming of Non-Terminally Differentiated B Cells

Initial attempts failed to reprogram bone marrow-derived B cells andspleen B cells that had been cultured on irradiated feeder cells in ESmedia supplemented with LIF and Dox, as the cells died within five daysin culture. We reasoned that addition of cytokines might be necessary toallow for an initial proliferation of the B cells that would ensure asufficient number of cell divisions necessary to initiate epigeneticreprogramming by expression of the four factors. Therefore, we optimizedculture conditions that would support immature and mature B cell growthas well as that of ES cells to ensure viability during the reprogrammingprocess from B to iPS cells.

Cells were grown on OP9 bone marrow stromal cells in media supplementedwith LIF which is required for ES cell growth, with IL-7, SCF and Flt-3Lwhich support B cell development (Milne at al., 2004), and with IL-4,anti-CD40 and LPS which are important for proliferation of mature Bcells (Hayashi et al., 2005). In initial experiments we detectedAP-positive colonies in cultures of sorted Pre- and Pro-B cell subsetsderived from 8 week old adult chimera bone marrow after 14 days of Doxtreatment. Small flat colonies appeared 3 days after Dox induction thatsubsequently underwent robust expansion. Around day 11 after Doxinduction smooth ES-like small colonies embedded within the granulatedlarge colonies were observed which became Nanog-GFP+ at day 14. Colonieswere picked 14 days after Dox induction from 3 independent experimentsand grown on MEF feeders in ES media without Dox. Within 3 passages over90% of the picked colonies grew into homogenous ES-like Nanog-GFP+ iPScells. In the following we will refer to these cell lines as iB-iPScells (for iPS cells derived from “immature” non-fully differentiated Bcells including Pre- and Pro-B cells)

Genomic DNA harvested from established iB-iPS cell lines was analyzed byPCR for heavy and light chain rearrangements. We used previouslydescribed degenerate primers that recognize the majority ofrearrangements involving three major families of V segments of the heavychain locus (V_(H)Q52, V_(H)7183-DJ, V_(H)Gam3.8), D_(H)-J_(H) heavychain rearrangements, and Igκ and Igλ light chain rearrangements (Changat al., 1992; Cobaleda at al., 2007a) (Table 2). Representative celllines reprogrammed from the B220+c-Kit+ Pro-B cell subpopulation showedthat some iPS lines carried D_(H)-J_(H) rearrangements (lines #1, 2, 7,9), whereas others did not show evidence for any IgH rearrangements(lines #3, 4, 6), as would be expected for rearrangements in the donor Bcell subset at the Pro-B cell stage of development. Cell linesestablished from the adult bone marrow-derived B220+CD25+ Pre-B cellscarried at least one V_(H)-DJ_(H) rearrangement and an additionalD_(H)-J_(H) or V_(H)-DJ_(H) rearrangement (lines #5, 8), both geneticrearrangements of the IgH locus typically observed in such B cellpopulations (Jung at al., 2006). IgH rearrangements in the iB-iPS wereverified by Southern blot analysis.

For subsequent analysis, we focused on cell lines that contained geneticevidence for IgH rearrangements, as only those can be definitivelytraced to cells committed to the B cell lineage. All iB-iPS cell linesstained positive for the ES markers AP, SSEA1 and Oct4, and all celllines tested (#5, 7, 8, 9) generated differentiated teratomas wheninjected into immunodeficient mice. Furthermore, we obtained adultchimeras from several iB-iPS cell lines (Table 1). RepresentativeSouthern blots of tail DNA from an iB-iPS#8 cell line-derived chimerashowed a heavy chain rearrangement pattern identical to the donor iB-iPScell line, thus confirming that the chimera was derived from therespective iB-iPS cell line and not from contaminating ES- orMEF-derived iPS cells. A chimera derived from iB-iPS line #9 produced100% germline transmission as demonstrated by the agouti coat color ofall mice obtained. As expected, Southern blot analysis confirmedsegregation of the rearranged IgH allele found in the donor iB-iPS linein some of the mice. These results demonstrate that cells committed tothe B cell lineage carrying D_(H)-J_(H) or V_(H)-DJ_(H) rearrangements,although not fully differentiated, can be reprogrammed to a pluripotentES-like state by the induction of the 4 transcription factors Oct4,Sox2, Klf4 and c-Myc.

Reprogramming of Terminally Differentiated B Cells

We failed to generate any reprogrammed AP+ colonies from mature spleenIgM+IgD+ cells or bone marrow derived IgK+ cells in 5 independentexperiments. This was puzzling given that IgM+IgD+ mature transgenic Bcells could be maintained in our culture conditions for up to 6 weeksand continued to express B cell markers. It appeared possible that thetransgenic B cells were able to proliferate in conditioned media withDox for a relatively extended period due to induction of c-Myc, which isknown to promote B cell growth and is a key player in B celltransformation (Zhu et al., 2005). We tested, therefore, the hypothesisthat additional pluripotency factors might be needed to achievereprogramming of mature B cells. Adult IgM+IgD+ spleen B cells wereinfected with combinations of retroviruses encoding 20 differentpluripotency factors that were originally generated to screen forfibroblast reprogramming (Takahashi and Yamanaka, 2006). Yet, theseexperiments repeatedly yielded negative results.

As an alternative approach, we aimed to “sensitize” the B cells torespond to Dox-dependent 4-factor induction by altering their mature Bcell identity. It has been shown that over-expression of the myeloidtranscription factor CCAAT/enhancer-binding protein-α (C/EBPα) is ableto reprogram B cells into macrophage-like cells (Xie et al., 2004) bydisrupting the function of Pax5, a transcription factor that is a masterregulator of mature B cell development and immunological function(Cobaleda et al., 2007b). In these experiments the C/EBPα transduced Bcells had been grown on bone marrow stromal cells in the presence ofmyeloid cytokines and had differentiated into functional macrophages(Xie at al., 2004). We tested, therefore, whether transduction withC/EBPα would facilitate reprogramming of mature B cells.

Adult spleen B cells derived from 10 week-old chimeras were transducedwith a retrovirus encoding C/EBPα and/or the IL7-Rα subunit and culturedon OP9 cells in the presence of Dox to induce the four factors Oct4,Sox2, Klf4 and c-Myc. AP positive colonies appeared after 14 days inculture in cells transduced with C/EBPα or with C/EBPα and IL7-Rα butnot in cells transduced with IL7-Rα alone. After 3 days of growth onOP9, small adherent colonies were formed which continued to grow intodenser granulated colonies. Similarly to Dox-induced Pre- and Pro-B cellcultures, small round ES-like colonies appeared within the large densegranulated colonies, and Nanog-GFP+ foci were readily detected atapproximately day 14.

Plating on OP9 bone marrow stromal cells was critical for recovering iPScells, as no iPS cells were detected when the cells were cultured on MEFfeeders or gelatin coated plates. Colonies isolated at day 14 werepassaged on MEF feeder cells without hematopoietic cytokines or Dox andwithin 3 passages all lines assumed an ES-like morphology and werepositive for the Nanog-GFP marker.

We performed FACS analysis to measure kinetics of SSEA1 and Nanogpluripotency marker activation in Dox induced bone marrow B220+ B cellpopulations and mature spleen IgM+IgD+ B cells infected with C/EBPαretrovirus. This assay showed similar reprogramming kinetics in whichSSEA1+ cells were initially detected at day 7 and became abundant at theday 11 after Dox addition. Nanog expression was detected at day 15similar to the sequential appearance of pluripotency markers duringreprogramming of MEFs (Brambrink et al., 2008). Our results suggest thattransduction with C/EBPα can sensitize mature B cells to respond to theexpression of Oct4, Sox2, c-Myc and Klf4 and re-express pluripotencymarkers.

We established 120 independent iPS lines that were picked fromindependent tissue culture wells containing IgM+IgD+ B cells from adultspleen and lymph nodes at 14 days after Dox addition and C/EBPαtransduction, and 9 cell lines were randomly selected for in depthcharacterization (Lines 1-6 obtained from adult spleen and 7-9 fromadult lymph nodes). In the following we will refer to these cell linesas “B-iPS” cells (iPS cells derived from mature “B” cells).

Next, we characterized marker expression, DNA methylation and histonemarks of the B-iPS cell lines. Immunoflorescence staining showed thatall B-iPS cell lines uniformly expressed ES cell markers AP, SSEA1antigen, Oct4 protein and were positive for Nanog-GFP. Gene expressionanalysis by RT-PCR showed that B-iPS and ES cells, but not primary Bcells, expressed comparable levels of Nanog, Ecat1, Rex1, Zfp296 andGDF3 genes. Bisulphite sequencing was performed to determine themethylation status of Oct4 and Nanog gene promoters for iB-iPS and B-iPScell lines. As expected, fibroblast and B cell control samples displayedextensive methylation at both promoters, whereas B-iPS and iB-iPS linesshowed widespread demethylation of these regions similar to that seen inES cells.

To assess the chromatin state of the cells, chromatinimmunopercipitation (ChIP) and real time PCR were performed to quantify‘active’ histone H3 lysine 4 trimethylation (H3K4me3) and ‘repressive’histone H3 lysine 27 trimethylation (H3K27me3) methylation marks on aselected set of genes known to be bivalent (carry both active andrepressive methylation marks) in ES cells (Bernstein et al., 2006). Ascells differentiate, such genes can become) “monovalent” and carryeither H3K4me3 or H3K27me3 marks, depending on their expression. Thepromoter region for the B cell transcription factor gene Pax5 displayedstrong enrichment for H3K4me3 methylation in the donor mature B cells,whereas H3K27me3 methylation predominated at the silent genes Zfpm2 andIrx2. Conversely, in B-iPS and ES cells all these genes carry equivalentenrichment for both histone modifications, consistent with the notionthat these bivalent domains were re-established during reprogramming.

In summary, our results indicate that the chromatin configuration of theB-iPS cells had been converted from a configuration typical ofterminally differentiated adult mature B cells to one that ischaracteristic for ES cells (Bernstein et al., 2006).

Rearrangements of Immunoglobulin Loci in B-iPS Cells Confirm Mature BCell Identity of the Donor Cells

In order to characterize the genomic rearrangements of the Ig loci inthe B-iPS cells, genomic DNA from MEF-depleted iPS cell lines grown ongelatin was analyzed for IgH, Igκ and Igλ rearrangements bycomplementary approaches that included Southern blotting, PCR andsequencing of individual PCR fragments (Alt et al., 1981; Chang et al.,1992; Cobaleda et al., 2007a; Lewis at al., 1982; Schlissel et al.,1991) (Table 6). All cell lines contained 2 heavy chain rearrangements:one was a productive in-frame V-DJ rearrangement whereas the other waseither a frozen D-J rearrangement or a non-productive V-DJrearrangement. These results are consistent with the well establishedobservation that adult mature B cells in the periphery have 2 rearrangedheavy chain loci (Jung et al., 2006).

As predicted for mature B cells, the light chain loci had one productivein-frame Igκ or Igλ light chain rearrangement (Jung et al., 2006).Though 95% of Igλ+ B cells in mice are known to carry unproductive Igκrearrangements, B-iPS cell line #9 was derived from a minor B cellsubpopulation with a rearranged productive Igλ chain and kappa locusthat was retained in the germline configuration (Nadel et al., 1990;Oberdoerffer et al., 2003). Finally, sequences obtained from heavy andlight chain rearrangements from B-iPS cell line #4 provided conclusiveevidence that the donor B cell nucleus that yielded this cell line hadundergone somatic hypermutation, a process that occurs after antigenencounter in vivo and involves acquiring a high rate of somaticmutations at “hotspots” located throughout the DNA encoding theimmunoglobulin variable region (Teng and Papavasiliou, 2007). Thisdirected hypermutation allows for the selection of B cells that expressimmunoglobulin receptors possessing an enhanced ability to recognize andbind a specific foreign antigen. The abundance of mostly non-silentmutations in the variable region of the productive rearrangements inthis cell line shows that non-naïve B cells that have alreadyencountered antigen in vivo are also amenable to direct reprogramming.

B-iPS#4 cell line likely arose from a contaminating IgM+IgD− cell duringthe cell sorting process because IgM+IgD+ B cells had been selected forreprogramming and this selection would be expected to yield only naïvemature B cells as cells that undergo antigen encounter and somatichypermutation downregulate the IgD antigen (Matthias and Rolink, 2005),Finally, the C/EBPα viral transgene was detected in genomic DNA from allB-iPS cell lines analyzed. The genomic analyses described above provideunequivocal evidence that the iPS cell lines were derived fromterminally differentiated adult mature B cells which had completed theirmaturation in the bone marrow, carried the expected functional heavy andlight chain rearrangements, and populated peripheral lymphoid organs.

Developmental Potential of B-iPS Cells

As an initial test for developmental potency we injected 8 B-iPS celllines subcutaneously into the dorsal flanks of immunodeficient (SCID)mice. Six weeks after injection, macroscopic teratomas were observed inall injected mice. Histological examination showed that the teratomascontained cell types representing all three embryonic germ layers,including gut-like epithelial tissues (endoderm), striated muscle(mesoderm), cartilage (mesoderm), neural tissues (ectoderm), andkeratin-containing epidermal tissues (ectoderm). To assess morestringently their developmental potential, individual B-iPS cell lineswere injected into diploid (2N) blastocysts resulting in the generationof viable, high-contribution chimeras from all 4 B-iPS cell lines tested(Table 5). Southern blot analysis of genomic DNA isolated from B-iPS #4-and #1-derived chimeras revealed the presence of genomic fragmentscorresponding to rearranged Igκ alleles identical to those observed inthe donor injected B-iPS cell lines. Importantly, B-iPS line #1contributed to the germline as was evident by the derivation ofoffspring carrying a constitutively expressed lentiviral transgene EGFPvector that was used for transducing B-iPS line #1 prior to blastocystinjections.

The generation of mice by tetraploid complementation, which involvesinjection of pluripotent cells in 4N host blastocysts, represents themost rigorous test for developmental potency because the resultingembryos are derived only from injected donor cells (Eggan et al., 2001).Both B-iPS lines tested (#4 and #9) were able to generate mid- andlate-gestation all B-iPS embryos' after injection into 4N blastocysts.Sensitive PCR analysis for the detection of a 2 Kb germ-line region fromthe B cell receptor heavy chain locus that is lost upon initiation ofgenetic rearrangement (Chang et al., 1992) shows that genomic DNA fromB-iPS #4 cell line embryos derived by tetraploid complementation hadlost the germ line band and carried only the D-J rearrangement aspredicted from the repertoire in the donor nucleus. This conclusion wasconfirmed by Southern blot analysis of genomic DNA from a day E14.5tetraploid embryo obtained from B-iPS#4 demonstrating two rearranged IgKlocus alleles, without any evidence for a germline allele. This is incontrast to DNA obtained from 2N chimeras that yielded the same 2rearranged IgK alleles and a germline band originating from hostblastocyst derived cells.

We next tested the ability of reprogrammed mature B cells to generatemonoclonal B cells in vivo as a result of the restrictions imposed bytheir pre-rearranged IgH and IgL loci (Hochedlinger and Jaenisch, 2002;Inoue et al., 2005; Oberdoerffer at al., 2003). To facilitate theisolation of B-iPS derived cells in chimeric mice, B-iPS lines #4 and 9were labeled with the GFP marker by lentiviral vector-mediatedtransduction prior to blastocyst injection. Surface expression of Igκand Igλ light chain proteins expressed on CD19+ cells purified fromperipheral blood was evaluated by FACS staining. All GFP+ B cells inB-iPS #4-derived chimeras expressed Igκ chain, but not Igλ protein,consistent with the genetic analysis that showed a functional Igκ lightchain rearrangement in this cell line. In contrast, B-iPS #9 cellline-derived B cells carried only a functional IgA light chainrearrangement.

Finally, we established two B-iPS cell lines that were generated bydirect infection of genetically unmodified mature B cells with theOct-4, Klf4, Sox2, c-Myc and C/EBPα grown in the same culture conditionsdescribed in our study and were capable of generating adult chimeras. Insummary, our results provide unequivocal molecular and functional proofthat mature B cell donor nuclei that contain functional light and heavychain rearrangements were reprogrammed to pluripotency. The cell linescarried productive heavy and light chain rearrangements, expressedpluripotency markers, generated live chimeras and contributed to thegerm line.

Efficiency of Reprogramming Mature Adult B Cells to Pluripotency

To estimate the efficiency of reprogramming of mature adult B cells topluripotency, a large starting pool (3*10^(^6)) of CD19+ B cellsisolated from the spleen of adult chimeras was infected with a C/EBPαencoding retrovirus carrying a neomycin resistance gene. After 24 hours,IgM+IgD+ B cells were plated as single cells in 96-well plates on OP9stromal cells in cytokine conditioned medium in the presence of Dox andLIF. Five days after plating puromycin and neomycin were added to theculture medium in order to select for transgenic B cells that had alsobeen infected with C/EBPα (FIG. 4). At day 20 wells that showed cellgrowth were screened by FACS for detection of Nanog-GFP+ cells. Wellsthat scored positive were expanded, and Nanog-GFP iPS cells appearedwithin 3 passages. PCR analysis of B-iPS lines obtained confirmed thatall cell lines obtained from two independent experiments originated fromC/EBPα-infected mature B cells that had distinct B cell receptorrearrangements. Based on these data, we were able to calculate theefficiency of reprogramming by dividing the number of GFP+ wellsobtained (output) by the number of C/EBPα-infected transgenic Bcell-containing wells (puromycin and neomycin double resistantwells=input). This calculation suggested that the relative efficiencyfor direct reprogramming of mature B cells was approximately 1 in 27-34cells. We attribute the relatively high efficiency of reprogramming tothe strong Dox-mediated induction of 4 out of the 5 ectopicallyexpressed factors that did not rely on retroviral vector infection andrandom proviral integrations.

TABLE 1 Summary of primary iPS lines. Initial Growth outgrowth GFP postprimary GFP (day picked) expression picking iPS line positive 1 (11) − +− — 2 (11) − + − — 3 (11) − − 4 (11) − − 5 (11) − + − — 6 (11) − + − — 7(11) − + + 18 8 (11) − + − — 9 (16) − + + 20 10 (16) − + + 20 11(16) + + + 18 12 (16) − − 13 (16) − + − — 14 (16) − + + 26 15 (16) − + −— 16 (16) − − 17 (16) − + − — 18 (16) − + − —

TABLE 2 Summary of secondary iPS lines. subclones Growth secondarypicked on GFP post iPS GFP day 16 expression picking line positive 1.1− + + 28 1.2 − + − — 1.3 − + + 30 4.1 − + − — 4.2 − − 4.3 − + − — 5.1− + + 32 5.2 − − 5.3 − − 6.1 − + −   —*** 6.2 − + −   —*** 6.3 − + −  —*** 8.1 − + + 28 8.2 − + + 36 8.3 − −

TABLE 3 Summary of genetically unmodified iPS derivation Picked #Prim/sec Background/type on day 16 expanded iPS lines 129/B6 F1/MEFs 8 32 129/B6 F1/TT 8 3 1 Balb/MEFs 8 3 2 B6/DBA F1/TT 8 3 1 Whole plate129/B6 F1/MBF — 5 5

TABLE 4 Summary of blastocyst injections. 2N injections 4N injectionsIn- Chime- In- Dead Live Cell jected Live rism jected embryos embryosline blast. chimeras (%) blast. (arrested) (analyzed) OG-7 25 2 15-60 744 (E11-15)* — OG-7.3 18 1 40 — — — OG-8.1 16 3 30-60 — — — OG-9 nd — —14 1 (E12.5)** — OG-10 18 3 20-40 — — — OG-14 nd — — 42 4 (E11-14)  3(E14.5) 129/B6 18   1*** *** — — — F1/MEFs Balb/c 22   3*** *** — — —MEFs

TABLE 5 Summary of blastocyst injections. The extent of chimerism wasestimated on the basis of coat color or EGFP expression. 2N injections4N injections Cell Injected Live Injected Dead embryos Live embryos lineblastocysts chimeras chimerism Germline blastocysts (arrested)(analyzed) iB-iPS 36 1 10-30 ND ND ND ND #1 iB-iPS 95 5 40-70 Yes ND NDND #4 iB-iPS 20 2 50-70 No ND ND ND #8 B-iPS 40 3 20-60 Yes ND ND ND #1B-iPS 24 2 30-50 No ND ND ND #2 B-iPS 135 6 30-80 ND 115  7 (E10-14.5) 3(E12.5) #4 2 (E14.5) B-iPS 95 8 30-80 ND 90 5 (E9-12.5)  5 (E12.5) #9B-iPS 46 3 30-60 ND ND ND ND #121 ND, not determined. 4N injectedblastocysts were analyzed between day E10.5 and E14.5. ‘Analyzed’indicates the day of embryonic development analyzed; ‘arrested’indicates the estimated stage of development of dead embryos.

TABLE 6 Primers used for PCR analysis of Ig rearrangements.K: G or T, M: A or C, S: C or G, R: A or G, W: A or T, Y: C or T.Sense Oligonucleotides Igh V_(H)J558 CGAGCTCTCCARCACAGCCTWCATGCARCTCARClocus (SEQ ID NO. 38) V_(H)7183 CGGTACCAAGAASAMCCTGTWCCTGCAAATGASC(SEQ ID NO. 39) V_(H)Q52 CGGTACCAGACTGARCATCASCAAGGACAAYTCC(SEQ ID NO. 40) V_(H)Gam3.8  CAAGGGACOGTTTGCCTTCTCTTTGGAA(SEQ ID NO. 41) DSF AGGGATCCTTGTGAAGGGATCTACTACTGTG (SEQ ID NO. 42) IgLVλ1 GCCATTTCCCCAGGCTGTTGTGACTCAGG loci (SEQ ID NO. 43) VκGGCTGCAGSTTCAGTGGCAGTGGRTCWGGRAC (SEQ ID NO. 44)Antisense Oligonucleotides Igh JH4 TCTCAGCCGGCTCCCTCAGGG locus(SEQ ID NO. 45) JH4 AAAGACCTGCAGAGGCCATTCTTACC (used with(SEQ ID NO. 46) DSF primer) IgL Jλ1,3 ACTCACCTAGGACAGTCAGCTTGGTTCC loci(SEQ ID NO. 47) Jκ5 ATGCGACGTCAACTGATAATGAGCCCTCTCC (SEQ ID NO. 48)

REFERENCES

The following references are cited herein and their teachings areincorporated by reference for all purposes.

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What is claimed is:
 1. A method of reprogramming a cell to a pluripotentstate comprising: (a) introducing into the cell exogenous factorscomprising (i) polynucleotides encoding Oct-4, Sox-2, and Klf-4, but notc-Myc; or (ii) Oct-4, Sox-2, and Klf-4 polypeptides, but not a c-Mycpolypeptide, and (b) culturing the cell for a sufficient amount of timeto reprogram the cell to the pluripotent state, wherein the reprogrammedcell does not express a selectable marker operably linked to apluripotency gene.
 2. The method of claim 1, wherein the polynucleotidesare introduced by transfection or viral infection.
 3. The method ofclaim 1, wherein step (a) comprises introducing said polynucleotidesinto the cell; and the method further comprising inactivating at leastone of the polynucleotides after the cell obtains the pluripotent state,wherein said inactivating optionally comprises excising at least aportion of a coding sequence from the at least one of thepolynucleotides, wherein the portion of the coding sequence is flankedby sequences that are recognized by a site-specific recombinase, andwherein said excising comprises introducing or expressing thesite-specific recombinase in the cell after the cell obtains thepluripotent state, wherein the site-specific recombinase removes theportion of the coding sequence from the at least one of thepolynucleotides.
 4. A method of reprogramming a human adult somatic cellto a pluripotent state comprising the steps of: (a) introducing into ahuman adult somatic cell reprogramming agents that contribute toreprogramming of the cell, wherein the reprogramming agents compriseOct-4, Sox-2, and Klf-4, but not c-Myc; and (b) culturing the cell in amedium comprising cell proliferation factors and factors that supportreprogramming for a period of time sufficient to reprogram the cell tothe pluripotent state, wherein the cell does not comprise a selectablemarker operably linked to an endogenous pluripotency gene.
 5. The methodof claim 4, wherein the reprogramming agents are introduced as (i)polynucleotides encoding Oct-4, Sox-2, and Klf-4, but not c-Myc; or (ii)Oct-4, Sox-2, and Klf-4 polypeptides, but not a c-Myc polypeptide. 6.The method of claim 4, wherein the somatic cell is a partiallydifferentiated cell or a fully differentiated cell.
 7. A method ofgenerating a reprogrammed human pluripotent cell comprising: introducinginto a human somatic cell, exogenous polynucleotides encoding Oct-4,Sox-2, and Klf-4, but not c-Myc; or exogenous Oct-4, Sox-2, and Klf-4polypeptides, but not a c-Myc polypeptide, culturing said human somaticcell for a sufficient amount of time to obtain a reprogrammed humanpluripotent that (i) is resistant to DNA demethylation; (ii) expressesendogenous Oct-4; (iii) differentiates into tissues having thecharacteristics of endoderm, mesoderm, and ectoderm when injected intoSCID mice; and (iv) does not comprise a selectable marker operablylinked to an endogenous pluripotency gene.
 8. The method of claim 7,wherein the exogenously introduced polynucleotides are introduced intothe human somatic cell by transfection or viral infection.
 9. The methodof claim 7, wherein step (a) comprises introducing said polynucleotidesinto the human somatic cell; and the method further comprisinginactivating at least one of the polynucleotides after the human somaticcell becomes the reprogrammed human pluripotent cell, wherein saidinactivating optionally comprises excising at least a portion of acoding sequence from the at least one of the polynucleotides, whereinthe portion of the coding sequence is flanked by sequences that arerecognized by a site-specific recombinase, and wherein said excisingcomprises introducing or expressing the site-specific recombinase in thecell after the human somatic cell becomes the reprogrammed humanpluripotent cell, wherein the site-specific recombinase removes theportion of the coding sequence from the at least one of thepolynucleotides.